S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including T and B lymphocytes) in the bloodstream and inflamed tissues, making them an effective therapeutic concept for autoimmune disorders. Although the effect of S1P receptor modulators in reducing circulating lymphocytes is well documented, the precise molecular role of the S1P1 receptor on these cell types is not fully understood. In this study, the mode of action of cenerimod on human primary lymphocytes in different activation states was investigated focusing on their chemotactic behavior towards S1P in real-time, concomitant to S1P1 receptor expression and internalization dynamics. Here, we show that cenerimod effectively prevents T and B cell migration in a concentration-dependent manner. Interestingly, while T cell activation led to strong S1P1 re-expression and enhanced migration; in B cells, an enhanced migration capacity and S1P1 receptor surface expression was observed in an unstimulated state. Importantly, concomitant treatment with glucocorticoids (GCs), a frequently used treatment for autoimmune disorders, had no impact on the inhibitory activity of cenerimod on lymphocytes.Cenerimod is a potent, selective sphingosine 1-phosphate receptor 1 (S1P Each receptor subtype shows a distinct expression profile, spanning a wide range of different cell types and tissues, mainly associated with functions in the central nervous system, the vascular system and immune system [1 receptor plays a key role in the functioning of endothelial cells and lymphocytes. In the case of lymphocytes, the S1P1 receptor is crucial in orchestrating the egress of T and B cells from secondary lymphoid organs to the lymphatic system and blood by receptor-mediated chemotaxis towards an S1P concentration gradient which is highest in blood/lymph. Due to the high concentrations of S1P in blood and lymph, a rapid internalization and thereby desensitization of the S1P1 receptor occurs on lymphocytes present there. Within lymphoid organs and tissue, the presence of a degradative S1P-lyase maintains low S1P levels, allowing the re-expression of the receptor—the required precondition for subsequent lymphocyte egress, thereby closing the cycle of lymphocyte trafficking [Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that acts as a ligand on a group of five G-protein-coupled receptors S1Pfficking . 1 receptor-mediated chemotaxis has been implicated in a variety of immune-related diseases and has therefore become an attractive therapeutic target. Fingolimod (FTY720), a non-selective S1P receptor (S1PR) modulator, had been approved by the FDA for the treatment of relapsing multiple sclerosis (RMS) as the first drug with this mode of action. Subsequently, various other selective S1PR modulators followed in the area of MS  and ulcerative colitis . These modulators act as functional antagonists on the S1P1 receptor by leading to sustained receptor internalization and desensitization. As a result, lymphocytes lose their ability to “sense” the S1P gradient, preventing their egress from the secondary lymphoid organs and leading to a decrease in lymphocyte counts, including pathogenic autoimmune cells in the bloodstream and in inflamed tissues [Due to its fundamental role in immune cell trafficking, S1P tissues ,4,5.1 receptor modulators [Systemic lupus erythematosus (SLE) is another indication for which this mode of action may be an appropriate means of intervention. This autoimmune disease with a high unmet medical need can affect virtually any organ of the body ranging from mild to life-threatening manifestations including irreversible organ(s) dysfunction and reduced life span. The incomplete understanding of SLE pathogenesis makes drug development in this disease particularly challenging. However, it has been shown that T and B lymphocytes play a major role in disease development via autoantibody production and tissue infiltration, making SLE an attractive target for S1Pdulators ,7.1 receptor modulator that has previously shown a dose-dependent reduction of circulating lymphocyte count and disease parameters in an SLE mouse model, as well as a dose-dependent reduction of lymphocyte count in patients with SLE [Cenerimod is a novel, potent, selective and orally active S1Pwith SLE ,9,10. Cu1 receptor modulators has been well characterized [1 receptor-mediated chemotaxis of lymphocytes towards S1P and the inhibitory effect of S1P1 receptor modulators on this process has only been partially described, especially for primary human cells.Although the effective reduction of peripheral T and B lymphocytes by cenerimod and other S1Pcterized ,9,10,11,1 receptor expression and internalization dynamics of primary human T and B lymphocytes of healthy donors in different activation states. In addition, S1P-mediated chemotaxis was analyzed using a novel real-time migration assay with subsequent characterization of the effects of cenerimod in this experimental setup. As GCs are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GCs on S1P1 receptor expression and function was additionally investigated.In this study, we characterized in vitro S1P1 receptor surface expression and internalization dynamics were characterized using flow cytometry. Primary human T cells were isolated from healthy donors and treated with different concentrations of S1P or cenerimod. Importantly, all experiments studying S1P1 receptor expression were performed using media lacking fetal calf serum (FCS), which is known to contain S1P, inducing unwanted immediate receptor internalization [+ or CD8+ T cell subtypes, inducing receptor internalization at the same concentration-dependency , enabling the tracing of cell divisions by dye dilution. Labeled cells were stimulated for two days with anti-CD3 and anti-CD28 antibodies in the presence of S1P, cenerimod or DMSO control. Following activation, T cells were allowed to proliferate for two consecutive days, and S1P1 receptor and CD69 surface expression throughout the different cell generations was tracked using flow cytometry. As seen in 1 receptor was almost absent on the cell surface upon initial activation and started to be re-expressed from 2nd–3rd generation in parallel with declining CD69 levels, showing the strongest expression in those cells that proliferated the most  were compared to a non-activated control. From Day 0 to Day 4 post activation, flow cytometry was used to analyze S1P1 receptor and CD69 surface expression throughout the different cell generations and time. As indicated in As previously reported for T cells, the activation of B cells leads to an upregulation of CD69 and the reciprocal downregulation of the S1P in mice . However1 receptor surface expression was immediately downregulated on Day 1 post activation, with a more pronounced effect by triple activation compared to CD40L alone  when analyzing the total protein expression levels in cell lysates by Western Blot  induced by both CD40L alone and more pronounced with triple activation. CD69 levels slowly decreased over time, but in contrast to observations in T cells, surface expression levels stayed relatively high throughout the entire experiment and remained higher than levels prior to activation. S1P0L alone B,C. A moanalyzed B,C, in cern Blot D. As seen number .1 receptor, as well as induction of proliferation by the strongest activation treatment. However, in contrast to the observations made in T lymphocytes, S1P1 receptor re-expression was induced even with clearly detectable CD69 surface expression. Furthermore, total S1P1 receptor expression, in contrast to surface expression, was clearly induced by B cell activation. In summary, B cell activation led to the expected upregulation of the activation marker CD69 and downregulation S1P1 receptor modulators using long-term, real-time assays in primary human cells have not been reported [In vitro data directly showing S1P-mediated B cell migration and its regulation by S1Preported ,26. TherNon-activated B cells effectively migrated towards S1P in a concentration-dependent manner, with the same optimal concentration range (80 nM) as observed for activated T cells. At concentrations of S1P above 400 nM, directed migration decreased concentration-dependently, resulting in a bell-shaped migration curve similar to that observed with T cells. Activated B cells migrated poorly towards S1P, but within the same concentration range as non-activated cells . CenerimTo study the functional antagonism of cenerimod on S1P-mediated B cell migration, S1P at an optimal concentration of 80 nM was applied as a chemoattractant to non-activated B cells, which showed a stronger migratory behavior. Before S1P application, cells were pretreated with different cenerimod concentrations. As observed for T lymphocytes, cenerimod effectively inhibited S1P-mediated migration in a concentration-dependent manner .Taken together, S1P acts as an efficient chemoattractant in primary human B lymphocytes with an optimal concentration response between 80 nM–400 nM. Interestingly in contrast to the observations made in T lymphocytes, more B cells migrated in a non-activated state compared to after cell activation. Contrary to S1P, cenerimod did not act as a chemoattractant, but efficiently prevented S1P-mediated migration in a concentration-dependent manner.To test whether the use of GCs could have an impact on the mode of action of cenerimod in B cells, prevention of S1P-mediated primary B cell migration was performed in the presence of prednisolone. B lymphocytes were pretreated with prednisolone or solvent control before cenerimod was added at different concentrations. After cenerimod pre-incubation, S1P (80 nM) was added as the chemoattractant. The results in 1 receptor modulators have shown their therapeutic potential in autoimmune disorders such as RMS and UC [1 receptor modulators can be efficacious therapies in certain autoimmune diseases by acting as functional antagonists on the S1P1 receptor, resulting in the trapping of autoreactive lymphocytes within the secondary lymphoid organs. Therefore, this mechanism of action may be applicable for the treatment of other autoimmune-related diseases with a high unmet medical need. This concept of interfering with the trafficking of lymphocytes has particular relevance for the treatment of SLE, a complex autoimmune disease with substantial involvement of autoreactive T and B cells in disease development [S1PS and UC . These eelopment ,7.1 receptor modulator has shown promising results in a relevant SLE mouse model as well as in a proof-of-concept study in patients with SLE, suggesting that cenerimod has the potential to treat patients with SLE [Cenerimod, a novel, selective S1Pwith SLE ,9,10. In1 receptor expression and S1P-mediated cell migration was studied in detail.In this study, the molecular mechanism of cenerimod acting on primary human T and B lymphocytes in the context of S1P1 receptor modulators is the effective induction of receptor internalization and desensitization after receptor engagement. Therefore, we first characterized S1P1 receptor expression on human T and B cells upon cenerimod treatment. For both cell types, cenerimod induced concentration-dependent internalization of the receptor similar to the natural ligand . With this set of data, we could show that cell activation can increase S1P-mediated lymphocyte migration as seen in the case of T cells. However, the timeframe after activation defining the S1P1 receptor surface expression status is crucial. As such, shortly after activation, where receptor re-expression has not yet occurred, T cell migration is diminished as reported by Graeler et al. [1 receptor surface expression show a stronger migrational behavior [1 receptor surface re-expression levels following activation did not exceed S1P1 receptor levels on the surface of non-activated cells, most likely explaining their reduced migrational activity. However, it cannot be excluded that receptor cell surface re-expression dynamics in B cells take longer than four days post activation—the experimental timeframe could not be extended due to cell viability in vitro, which was lower for activated B cells compared to activated T cells in the tested conditions. Nevertheless, as shown by Western blotting, a strong induction of total S1P1 receptor protein production was detected in activated B cells at Day 4, which did not result in increased cell surface expression. The biological significance of this observation would need further investigation.We also tested lymphocyte migration in the context of different cell activation states. Consistent with the previous characterization of S1Pr et al. . At latebehavior . For B c1 receptor modulators in autoimmune diseases is to prevent the circulation of autoreactive lymphocytes. Importantly, cenerimod was able to efficiently prevent S1P-mediated migration for both T and B cells in a concentration-dependent manner. With cenerimod acting as a selective S1P1 receptor modulator, this study confirms the S1P1 receptor as the responsible S1P receptor in lymphocyte chemotaxis towards S1P in this context [As stated previously, a key rationale of the application of S1P context ,19. Thes1 receptor modulator cenerimod in this biological context, taking one more step in tackling the treatment of autoimmune diseases.In summary, we were able to further elucidate the underlying molecular principles of human T and B cell migration towards the chemoattractant S1P and confirm the effectiveness and mode of action of the novel S1P1 receptor in vitro as in-house data have shown that lymphocytes isolated with MACSxpress preserve S1P1 receptor surface expression in contrast to density gradient isolation. Cells were taken up in OpTmizer T Cell Expansion Medium  containing the corresponding supplement, 2 mM L-glutamine and penicillin–streptomycin (P/S). Importantly, all experiments studying S1P1 receptor expression were performed using this special OpTmizer medium, lacking FCS, which is known to potentially contain S1PR ligands, inducing unwanted immediate receptor internalization. Therefore, cells were cultured overnight to allow S1P1 receptor surface re-expression. Cenerimod and S1P  were added in serial dilution to cells and incubated at 37 °C for 30 min/24 h. For testing the effect of GCs, prednisolone (100 nM) was preincubated before ligand addition for another 30 min at 37 °C. Then cells were washed once and subsequently stained using the following antibodies: CD3BD Horizon™ V450 , CD4Brilliant Violet 785™ , CD19Brilliant Violet 510™ , S1P1 receptoreFluor660 . Every condition was performed with technical replicates. One representative experiment (n = 3) from one healthy donor is shown per figure. Samples were measured using NovoCyte/NovoCyte Quanteon ) and subsequently analyzed by Flowjo software (Becton Dickinson) and Prism (graphpad).Blood from healthy donors was collected in EDTA blood collection tubes, and T and B lymphocytes were isolated using MACSxpress Cell Isolation Kits . The choice of isolation method is crucial for studying S1PTM according to manufacturer’s protocol , which were removed after 48 h/72 h, and replaced by fresh media, containing IL-2 ) to promote T cell survival and proliferation. B cells were either activated by CD40L ) alone or in combination with IL-21 ) and F(ab’)2-Goat anti-Human IgG, IgM (H+L) Secondary Antibody ). For co-treatment with S1P or cenerimod, application occurred right after CTV labeling and S1P or cenerimod addition was refreshed after 48 h. Every 24 h, one fraction of cells was washed and stained with the following antibodies for flow cytometry analysis: CD69Alexa Fluor 488 /CD69PE-Vio770 , S1P1 receptoreFluor660 , CD19PE , CD4PE . Every condition was performed with technical replicates. One representative experiment from one healthy donor (n = 3) is shown per figure. Samples were measured using NovoCyte/NovoCyte Quanteon (ACEA Biosciences Inc.) and subsequently analyzed by Flowjo software (Becton Dickinson) and prism (graphpad). B and T lymphocytes were isolated and taken up in OpTmizer T Cell Expansion Medium as described above. Cells were then labeled with CellTrace™ Violet (CTV) cell proliferation Kit according to the manufacturer’s protocol . Cells were left untreated or activated as follows: T cells were activated by adding human T activator CD3/CD28 Dynabeadsn = 3) is shown per figure. Cell migration was measured by taking whole-well images in a 2 h or 3 h time-lapse, using the IncuCyte® ZOOM/SX5 (Sartorius), allowing vertical-directed cell migration to be quantified by the decrease of cell count on the top of the insert membrane over time. Data were analyzed using IncuCyte® ZOOM 2016B software (Sartorius) and prism (graphpad).T and B lymphocytes were isolated and taken up in OpTmizer T Cell Expansion Medium as described above. Cells were left untreated or activated as described in ® Turbo™ Transfer System. Proteins of interest were detected using the following antibodies: anti-S1P1 receptor  anti-Actin , diluted in 3% milk/PBS–Tween (0.1%). One representative experiment from one healthy donor (n = 3) is shown per figure.B lymphocytes were isolated and activated as described above. Cells were washed with PBS before the addition of lysis buffer , phosphatase inhibitor cocktail  followed incubation on ice (10 min). Lysates were cleared by centrifugation and taken up in NuPage LDS buffer  together with 20 mM DTT. Proteins were separated on 4–20% Criterion™ TGX Stain-Free™ Protein Gel , using corresponding running buffer Tris/Glycine/SDS . Subsequent immunoblotting on PVDF membranes  was performed using the Biorad Trans-Blotn = 3 using material from different human healthy donors. Statistical analysis was performed using Prism version 8.1.1 . The Kruskal–Wallis nonparametric test was applied following post-hoc analysis using Dunn’s test to compare each group to solvent control. p-values were adjusted for multiple comparisons. Differences were considered significant at p-values < 0.05.All data are expressed as mean of technical replicates ± standard error of the mean (SEM). Each experiment was performed


TNNI3K) on sepsis-induced myocardial dysfunction (SIMD) and further explore the underlying molecular mechanisms. In this study, a lipopolysaccharide- (LPS-) induced myocardial injury model was used. qRT-PCR was performed to detect the mRNA expression of TNNI3K. Western blot was conducted to quantitatively detect the expression of TNNI3K and apoptosis-related proteins . ELISA was performed to detect the content of lactate dehydrogenase (LDH) and creatine kinase (CK). TUNEL assay was used to detect the apoptosis of H9C2 cells. In LPS-induced H9C2 cells, TNNI3K was up regulated. Besides, the CK activity, the content of LDH, and the apoptosis of H9C2 cells were significantly increased after treatment with LPS. Silencing TNNI3K decreased the LDH release activity and CK activity and inhibited apoptosis of H9C2 cell. Further research illustrated that si-TNNI3K promoted the protein expression of Bcl-2 and decreased the protein expression of Bax and cleaved caspase-3. The study concluded that TNNI3K was upregulated in LPS-induced H9C2 cells. Importantly, functional research findings indicated that silencing TNNI3K alleviated LPS-induced H9C2 cell injury by regulating apoptosis-related proteins.The aim of this study was to investigate the effect of cardiac troponin I-interacting kinase ( Sepsis can lead to multiorgan dysfunction, among which sepsis-induced myocardial dysfunction (SIMD) is a serious complication . The iniDespite years of research, the mechanism by which sepsis induces SIMD is complex and controversial . At presCardiac troponin I interaction kinase (TNNI3K) is a member of the mitogen-activated protein kinase (MAPKKK) family and cardiac anchor protein repeat kinase (CARK), which was originally cloned by researchers of Peking University Union Medical College in 2003 . TNNI3K Herein, the aim of this study was to establish a rat model of sepsis treatment with LPS to investigate the effect of TNNI3K on SIMD.The rat myocardial cells (H9C2) were obtained from American Type Culture Collection . After resuscitation, they were cultured in RPMI-1640 medium containing 10% fetal bovine serum  in a humidified environment. When cells attained 70% to 80% confluences, transfection experiments could be conducted.μg/mL LPS for 24 h. To detect the effect of TNNI3K on myocardial injury, the cells were divided into six groups: (1) control group, treatment with 10% FBS; (2) LPS group, treatment with 10 μg/mL LPS for 24 h; (3) si-NC group, cotransfection with si-NC; (4) si-NC+LPS group, cotransfection with si-NC and LPS; (5) si-TNNI3K, cotransfection with si-TNNI3K; and (6) si-TNNI3K+LPS group, cotransfection with si-TNNI3K and LPS. This study was approved by the Ethics Committee of the Third Hospital of Hebei Medical University.To establish a model of myocardial injury, H9C2 cells were divided into two groups: (1) control group, treatment with 10% FBS, and (2) LPS group, treatment with 10 μg/mL LPS cells were added for 24 h.si-NC and si-TNNI3K were constructed by Ribobio Corporation , and plasmid dosage per transfection was 100 ng. The cells were divided into groups according to the experimental plan and inoculated into 6-well plates. Lentivirus infection was carried out after the cells adhered to the wall. After 24 h, 10 ΔΔCt- methods represented the fold changes of gene expression. The primer sequences for this experiment are shown below: TNNI3K sense (CGATACCGAGCCAACACCTA) and antisense (GCACCCACAAACTGAACCAC) and β-actin sense (GGAGATTACTGCCCTGGCTCCTAGC) and antisense (TGGCCGGACTCATCGTACTCCTGCTT).TRIpure reagent  was used to isolate the total RNA from samples, and PrimeScript RT kit  was used for reverse transcription. After the sample was prepared, the expression level was detected with SYBR green, and GAPDH was controlled as the internal parameter. 2According to the manufacturer's instruction, the proteins were extracted and its concentration was measured. Subsequently, the prepared protein was separated by polyacrylamide-SDS gels and then transferred onto PVDF membranes . After blocking, the PVDF membrane was subjected to incubation with primary antibodies: TNNI3K , Bcl-2, Bax, and cleaved caspased-3 . On the following day, the membrane was incubated with the secondary antibody at 37°C for 45 min and the intensity of protein expression was detected by ECL chemiluminescence .Concentrations of CK in the LPS-induced H9C2 cells were assessed by ELISA using a commercially available ELISA kit  according to the manufacturer's instructions.4) were seeded into 96-well plates and cultured for 12 h. After being collected, the LDH release activity from damaged cells was measured using a Cytoxicity Detection Kit . The absorbance values at 490 nm were detected using a microplate reader (Bio-Rad).Briefly, cells (1 × 10μg/mL) at 37°C. Afterwards, H9C2 cells were incubated overnight with the two  TUNEL reagents. Finally, TUNEL staining was observed under an optical microscope .TUNEL detection kit  was used to detect the apoptosis of H9C2 cells. First, myocardial cells were fixed with 4% formaldehyde and then washed in PBS containing proteinase K . One-way ANOVA followed by Dunnett's multiple comparison was applied to assess the differences between the groups. P < 0.01). Similarly, in the translational level, LPS-induced H9C2 cells also showed a significant increase protein expression of TNNI3K (P < 0.01).To investigate the role of TNNI3K in myocardial injury in rats, we measured the expression of TNNI3K at both transcriptional and translational levels. As indicated in f TNNI3K . CorrespP < 0.01). To further verify this conclusion, LDH assay and TUNEL assay were conducted to detect the death status and apoptosis of the cells. In LDH assay, we found that the LDH release activity from H9C2 cells was remarkably increased, suggesting that LPS aggravated H9C2 cell injury . In Figures We have shown that silencing TNNI3K could alleviate LPS-induced H9C2 cell apoptosis. To further explore the deep mechanism of TNNI3K affecting myocardial injury, we examined the expression of apoptosis-related proteins. As depicted in SIMD is an important part of cardiovascular failure in septic shock, leading to high mortality and poor prognosis in patients with sepsis . In thisin vitro and in vivo model studies. For example, intraperitoneal injection of LPS can significantly increase the expression of inflammatory factors in myocardial tissue and cardiac dysfunction [LPS is the main component of gram-negative bacilli cell wall, evoking SIMD and organ injury . Studiesfunction  and LPS function . In thisTNNI3K is a cardiomyocyte-specific kinase. A previous study has shown that in a TNNI3K overexpression knockout mouse model, TNNI3K can increase the area of ischemic heart infarction, induce mitochondrial ROS generation, and cause cardiomyocyte mitochondrial dysfunction and bioenergy damage . TNNI3K It has been reported that the occurrence of SIMD was closely related to apoptosis . Bcl-2 iIn this study, we mainly explored the functionality of TNNI3K and its potential molecular mechanisms in SIMD. We found that TNNI3K was upregulated in LPS-induced H9C2 cell and silencing TNNI3K alleviated LPS-induced H9C2 cell injury by regulating apoptosis-related proteins. Collectively, these observations indicated that TNNI3K may be utilized as a novel target to treat SIMD .


Ligustrum witches’ broom phytoplasma (LiWBP). To gain additional molecular information on the 16S ribosomal RNA and 23S rRNA intergenic spacer region of LiWBP, not identified previously, the P1/P7-primed amplicons were also sequenced and analyzed. The results show that maize isolates from Turkey share 99.6% to 100% of identity among them, whereas the highest identity found (91%) was with members of groups 16SrII and 16SrXXV . This distant relationship between LiWBP and members of 16SrII and XXV was also confirmed by RFLP and phylogenetic analyses. This is the first finding of LiWBP on maize in nature, where it was found responsible for phyllody disease of corn plants in Turkey. The additional molecular information acquired in this study on the 16S–23S rRNA intergenic spacer region of LiWBP further corroborates its distant relationship to any other phytoplasma groups.In the 2019–2020 growing season, two corn fields located in İmamoğlu town  were surveyed following the appearance of phytoplasma-like symptoms on maize plants. A total of 40 samples were collected and tested in first-round and nested PCR using universal primer pairs P1/P7 and R16F2n/R16R2, respectively. All maize-diseased plants reacted positively, whilst no PCR amplifications were obtained from asymptomatic plants. Blast sequence analysis of R16F2n/R16R2-primed amplicons from different maize isolates showed 99.2% to 100% of identity with the 16S rRNA gene of  Zea mays L.) production occupies third place after wheat and barley in Turkey [Maize , previously named as Ligustrum ovalifolium phytoplasma [Blast nucleotide sequence analyses of twenty R16F2n/R16R2-primed PCR products, obtained from 10 infected plants/field, showed that the phytoplasma affecting maize plants is the toplasma , which dTo gain additional molecular information on the 16S rRNA and 23S rRNA intergenic spacer region of LiWBP, not identified previously, the P1/P7-primed PCR amplicons of LiWBP-infected maize samples were further sequenced. In total, 12 P1/P7-primed amplicons were observed in agarose gel electrophoresis, out of 30 generated from the use of R16F2n/R16R2 primers in PCR when applied on infected maize plants.The sequencing of all P1/P7-primed amplicons showed that four sequence types of LiWBP were present in maize-infected plants that shared 99.6% to 100% of identity among them. These isolates were denoted F1/4, F1/51, F2/10 and F2/57 and their sequences were deposited in GenBank with the accession numbers HG994077–HG994080, respectively. However, the Blast analysis showed that members of groups 16SrII and 16SrXXV  shared the highest identity (ca. 91%) with the LiWBP maize isolates.The virtual restriction fragment length polymorphism (RFLP) patterns derived from the 16S rDNA R16F2n/R16R2 fragments of LiWBP were different from the reference patterns of all previously established 16Sr groups/subgroups (data not shown), but not from LiWBP Fe1 and Fe2 isolates (accession numbers HE649494 and HE649495) previously identified in Ligustrum plants . AlthougIn the RFLP analysis, the similarity coefficient was 0.65, which is much lower than the conventional threshold (0.85) stabilized for the delineation of a new and distinct phytoplasma 16Sr subgroup lineage . This diLigustrum ovalifolium) and Black locust tree (Robinia pseudoacacia) [The search for additional phytoplasma-derived sequences different from those found for LiWBP that might be responsible for the disease on maize plants, by sequencing 42 PCR amplicons (30 R16F2n/R16R2- and 12 P1/P7-primed amplicons from infected plants) was unsuccessful, as all sequences found were from LiWBP. These data suggest that LiWBP is a unique pathogen responsible for the symptoms observed on maize-affected plants in the two visited fields. This result was also corroborated by the RFLP analyses (using AluI and HhaI endonucleases) performed on eight R16F2n/R16R2-primed amplicons from infected plants, which generated DNA profiles similar to those obtained from the virtual RFLP for each enzyme (data not shown). The lack of information on phytoplasma infections on maize crops in Turkey makes this study the first report on such infection, together with maize as a new host plant for LiWBP in nature, besides the ornamental California privet plant (oacacia) . The sevCandidatus Phytoplasma” species should refer to a single, unique 16S rRNA gene sequence (>1200 bp), and a strain can be recognized as a novel “Ca. Phytoplasma” species if its 16S rRNA gene sequence has <97.5% similarity to that of any previously described “Ca. Phytoplasma” species.The search for insect vectors of phytoplasmas, i.e., leafhoppers, planthoppers and psyllid, that might have inoculated LiWBP onto maize plants from other hosts, together with their incidence in maize crops, is ongoing in order to draw an epidemiological scenario of this new pathogen in Adana Province. The sequence information acquired in this study on the 16S–23S rRNA intergenic spacer region of LiWBP suggests its classification as a new taxon among phytoplasma groups, since the 91% of identity found between LiWBP and the closest members of phytoplasma groups (16SrII and 16SrXXV) is largely distant from the demarcation criteria recommended by the Phytoplasma/Spiroplasma Working Team Phytoplasma Taxonomy Group , accordiDuring May–July 2019–2020, several visits were conducted in two maize fields (F1 and F2) at İmamoğlu town to investigate the etiology of maize-diseased plants showing symptoms of phyllody and deformation of tassel of male inflorescence, formation of multiple empty corncobs and proliferation in female inflorescence resulting in silkless and empty corncobs, bushy growth and dwarfing . Twenty w/v cetyltrimethylammonium bromide, 1.4 M NaCl, 0.2% 2-β-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl, 2% polyvinylpyrrolidone, pH 8.0). Aliquots of 600 μL of the extract were incubated at 65 °C for 30 min. An equal volume of chloroform-isoamyl alcohol (24:1) was added to the sample and vigorously mixed and centrifuged at 12,000 rpm for 10 min. Further extraction was performed with chloroform-isoamyl alcohol (24:1) addition followed by centrifugation at 12,000 rpm for 10 min. The nucleic acid was precipitated overnight at −20 °C in isopropanol, and the pellet was washed with 70% ethanol, dried at room temperature, dissolved in 50 μL of sterile water and used as DNA template for PCR amplification.DNA was extracted from midrib tissues of leaves and tassels of symptomless and diseased plants showing different types of symptoms , accordiPolymerase chain reaction and nested PCR assays were performed using the universal phytoplasma primer pairs P1/P7 ,21 and RTM PCR Cloning vector pSC-A , subcloned into Escherichia coli DH5α or SoloPACK cells and sequenced from both forward and reverse plasmid (pUC18) sequencing primers (T3 and T7) . Four DNA clones from each PCR-positive sample were sequenced. The nucleotide sequence data were assembled by employing the Contig Assembling program of the sequence analysis software BIOEDIT, version 7.0.0 .The P1/P7- and R16F2n/R16R2-primed PCR products obtained from maize plants were cloned using StrataCloneSequences of the R16F2n/R16R2-primed PCR products obtained from diseased maize plants were subjected to virtual RFLP analysis using the iPhyClassifier tool . Each 16Candidatus Phytoplasma” species available in GenBank. The evolutionary distances were computed using the maximum composite likelihood method [A phylogenetic tree was constructed using the sequences of 29 “d method . The evod method .


Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020–2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu–Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia. Avian influenza virus (AIV) subtype A (H7) has raised global concerns because it has been a leading cause of zoonotic infections over the past two decades . In partIn early 2018, a severe H7N4 infection was confirmed in a 68-year-old woman in Jiangsu, China, which was the first human case of infection with H7N4 LPAIV ,10. PhylIn South Korea, nationwide AIV surveillance has been conducted to rapidly detect and respond to potential introductions and outbreaks of H5 and H7 LPAI and HPAI . During A total of 16,293 samples  were collected from major migratory habitats in South Korea between October 2020 and April 2021. Sample collection was conducted by the Livestock Health Control Association or by regional veterinary offices according to the national surveillance program in South Korea. Samples were examined by virus isolation in 9–11-day-old specific-pathogen-free (SPF) embryonated eggs. After incubation for 4 days at 37 °C, the eggs were chilled, and the allantoic fluid was harvested and determined by hemagglutination assay using chicken erythrocytes. Host-species identification was confirmed using a barcoding system utilizing mitochondrial DNA from feces, as previously described .https://www.ncbi.nlm.nih.gov/genomes/FLU, (accessed on 30 April 2021)) and GISAID EpiFlu ) databases, and used for comparative phylogenetic analysis.The whole genome was amplified, and next-generation sequencing was conducted using the Illumina MiSeq platform, as described previously . Trimmedhttps://beast.community/logcombiner (accessed on 30 April 2021)) after a 10 percent burn-in, yielding a total of 18,002 parameters and posterior trees. The parameters were analyzed with Tracer v1.7.2 (http://tree.bio.ed.ac.uk/software/tracer/ (accessed on 30 April 2021)), and all parameters had an effective sample size greater than 200. A maximum clade credibility (MCC) tree was generated using TreeAnnotator in BEAST and visualized using FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 30 April 2021)).The maximum likelihood (ML) phylogenetic trees for all genes were constructed with RAxML version 8.2.12 using a gamma distribution and a general time-reversible model with 1000 rapid bootstrap replicates . The genBetween October 2020 and April 2021, a total of 291 AIVs were isolated from wild bird habitats in South Korea. The proportion of H7 viruses isolated was 5.8% (17/291). In addition, no LPAIVs, except for subtype H9N2 viruses, were detected in commercial poultry under nationwide surveillance in the same period. In a preliminary phylogenetic analysis of the HA gene, we identified five H7 AIVs genetically similar to the Jiangsu–Cambodian H7 gene. The five H7 AIVs contained different neuraminidase (NA) subtypes, including an H7N8 and an H7N9 detected from Jeollabuk-do Province in November 2020, and two H7N3 and an H7N9 from Jeju Province and Chungcheongbuk-do Province from January to March 2021 . The hosPhylogenetic analysis showed that the HA gene of the H7 isolates from wild ducks in South Korea formed a monophyletic cluster, and the tMRCA was estimated to be 24 April 2019 (95% highest posterior density (HPD): from 16 March 2018–17 April 2020) . It sharThe phylogenies of NA genes indicated that all subtypes of the NA gene  of the isolates belonged to the Eurasian lineage . The N9 A genomic constellation analysis demonstrated that all H7 isolates from South Korea contained a mix of gene segments from different viruses , suggestThe amino acid sequence of the HA protein of all H7 isolates in this study contained a monobasic arginine residue at the cleavage site (PELPKGR/GLF), indicating a low-pathogenicity phenotype in chickens  Table 3Table 3. Recently reported human infection with the H7N4 LPAIV has raised concerns about the pandemic potential of the virus, even though there was no evidence that human-to-human transmission has occurred. Considering that the pathogenicity in mice of the H7N6 virus belonging to the Jiangsu–Cambodian cluster was similar to that of the Chinese H7N9 viruses , the ris


RAB7A (RAs-related in Brain 7A) is a master regulator of intracellular traffic controlling transport to late endosomes and lysosomes, two organelles of the endocytic pathway important for degradation. Thanks to this function, RAB7A is also involved in cellular processes linked to cancer, such as apoptosis, cytoskeletal reorganization, and cell migration. Therefore, the interest in the role of RAB7A in cancer progression is increasing. Previously, we demonstrated that RAB7A regulates phosphorylation and assembly of vimentin, a cytoskeletal intermediate filament protein, which is also an important mesenchymal marker of cancer cells. The aim of the present study is the identification of the kinases responsible for vimentin phosphorylation whose activity is affected by the modulation of RAB7A expression. We found that RAB7A is able to regulate AKT  and PAK1 (P21-Activated Kinase 1) and several of their downstream effectors, which control proliferation, apoptosis, survival, migration, and invasion. These data suggest that RAB7A could have a key role in cancer development.RAB7A is a small GTPase that controls the late endocytic pathway but also cell migration through RAC1 (Ras-related C3 botulinum toxin substrate 1) and vimentin. In fact, RAB7A regulates vimentin phosphorylation at different sites and vimentin assembly, and, in this study, we identified vimentin domains interacting with RAB7A. As several kinases could be responsible for vimentin phosphorylation, we investigated whether modulation of RAB7A expression affects the activity of these kinases. We discovered that RAB7A regulates AKT and PAK1, and we demonstrated that increased vimentin phosphorylation at Ser38 (Serine 38), observed upon RAB7A overexpression, is due to AKT activity. As AKT and PAK1 are key regulators of several cellular events, we investigated if RAB7A could have a role in these processes by modulating AKT and PAK1 activity. We found that RAB7A protein levels affected beta-catenin and caspase 9 expression. We also observed the downregulation of cofilin-1 and decreased matrix metalloproteinase 2 (MMP2) activity upon RAB7A silencing. Altogether these results demonstrate that RAB7A regulates AKT and PAK1 kinases, affecting their downstream effectors and the processes they regulate, suggesting that RAB7A could have a role in a number of cancer hallmarks. RAB (Ras-related in Brain) proteins are small GTPases that act as molecular switches to regulate many steps of membrane traffic, from the formation of vesicles at donor compartments to their transport, tethering, and fusion to the correct target compartments . RAB proTwo intermediate filament proteins, peripherin and vimentin interact with RAB7A, which regulates their assembly and phosphorylation state ,10. In pPhosphorylation of vimentin residues is mediated by several different kinases ,16,17,18In this study, we defined vimentin domains responsible for the interaction with RAB7A and we investigated the role of RAB7A on the activation state of several kinases that could, in turn, regulate vimentin phosphorylation. We established that RAB7A is able to regulate PAK1 abundance and AKT activation and that AKT could be the key element through which RAB7A modulates vimentin phosphorylation state and possibly cell adhesion.2. Cells were confirmed to be contamination-free, and their identity was confirmed by short-tandem repeat profiling.HeLa  and NCI H1299  cells were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS , 2 mM L-glutamine, 100 U/mL penicillin, and 10 mg/mL streptomycin in an incubator at 37 °C, under 5% COAKT inhibitor GDC-0068 was supplied by Cayman Chemical . Other chemicals were from Sigma-Aldrich . Tissue culture reagents were from Sigma-Aldrich or Gibco .Y183F and RAB7AY183H plasmids were obtained by PCR-mediated mutagenesis using the QuickChange XL Site-Directed Mutagenesis Kit . The mutagenesis was performed on RAB7A wildtype cDNA previously cloned in pcDNA3_2XHA, in frame with DNA coding for HA tag. Oligonucleotides used were 5′-GAAACAGAGGTGGAGCTGTTCAATGAATTCCCTGAACC-3′ and 5′-GGTTCAGGGAATTCATTGAACAGCTCCACCTCTGTTTC-3′ for RAB7AY183F and 5′-ACAGAGGTGGAGCTGCACAATGAATTCCCTG-3′ and 5′-CAGGGAATTCATTGTGCAGCTCCACCTCTGT-3′ for RAB7AY183H. For the luciferase assays, we used the vector pCEFL-AU5 RasV12 and a reporter plasmid containing five consensus NF-kB (Nuclear Factor Kappa B) responsive sites followed by the luciferase gene (Stratagene), as previously described [Plasmids encoding pcDNA3_2XHA and pcDNA3_2XHA-RAB7A wild type have been described previously . For theescribed . All thePrimary antibodies used in this work were rabbit polyclonal anti-HA (ab9110) from Abcam ; mouse monoclonal anti-RAB7A (sc376362), anti-vimentin (sc6260), anti-p-αPAK (sc-135755), anti-αPAK (sc-166887), anti-cofilin-1 (sc-53934), anti-caspase 9 (sc-56073), rabbit polyclonal anti-myc (sc-789), anti-p-AKT 1/2/3 (sc7985-R), anti-AKT 1/2/3 (sc8312), anti-p-PKA α/β/γ cat (sc32968), anti-PKA (sc903), and ROCK-2 (sc5561), from Santa Cruz Biotechnology ; mouse monoclonal anti-β-catenin (610154) from BD Transduction Laboratories ; rabbit polyclonal anti-cleaved caspase 9 (#7237), anti-NF-kB p65 (#8242), anti-matrix metalloproteinase 2 (MMP2) (#4022), anti-p-vimentin S38 (#13614), and anti-p-p65/RelA (Ser536) (#3033) from Cell Signaling Technology ; mouse monoclonal anti-tubulin (T5168) from Sigma-Aldrich and rabbit polyclonal p-ROCK2 (GTX122651) from GeneTex . Secondary antibodies conjugated to fluorochromes  or horseradish peroxidase  were from Invitrogen  or Santa Cruz Biotechnology .Transfection was performed using Metafectene Pro from Biontex  according to the protocol provided by the manufacturer or using Amaxa Cell Line Nucleofector Kit V  as previously described . Cells wFor co-immunoprecipitation experiments, cells were lysed in lysis buffer  and immunoprecipitation was performed using anti-HA resin , following the protocol provided by the manufacturer. Briefly, cell lysates were incubated at 4 °C with 25 µL of resin. After 1 h on a rotating wheel, immunoprecipitates were washed three times with washing buffer  and once with 10 mM Tris, pH 7.5, as previously described . FinallyPull-down experiments were performed as previously described . BrieflyCells were lysed with RIPA (Radioimmunoprecipitation Assay) buffer , 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) plus protease inhibitor cocktail  and phosphatase inhibitors (phosphatase inhibitor cocktail 1 from Sigma-Aldrich). Lysates were loaded onto SDS-PAGE and separated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane from Millipore  and probed with the primary antibody diluted in 2% milk in PBS  followed by HRP-conjugated secondary antibody, as previously described . Bands w® Ultra (Merck Millipore), quantified by Bradford assay and gelatin zymography was performed as previously described [2, 1 µM ZnCl2 to remove SDS and this step was followed by three more washes in distilled water. After washing, the gel was incubated at 37 °C for 24 h in activation buffer containing 1% Triton X-100, 50 mM Tris-HCl pH 7.5, 5 mM CaCl2, and 1 µM ZnCl2, and then was stained with 0.5% Coomassie Blue R-250  and destained in 40% Methanol and 10% acetic acid.HeLa cells were seeded, transfected with control RNA or RAB7A siRNA and the day after the replating, cells were cultured in serum-free medium for 48 h. Conditioned media were collected from cells, samples were concentrated using Amiconescribed . Briefly4Cl for 10 min and then incubated at room temperature with primary and secondary antibodies diluted in 0.1% saponin in PBS. After several washes, coverslips were mounted with mowiol and viewed with Zeiss LSM 700 confocal microscope.HeLa cells were fixed for 20 min in 3% paraformaldehyde and then permeabilized with 0.1% Triton X-100 in PBS. Subsequently, samples were quenched in NHhttps://www.rcsb.org/ accessed on 1 April 2021). To perform docking simulation two online servers PatchDock [Y183H we used a dynamic approach (docking-based). To estimate the dynamic feature of the protein we used CABSflex server using as input the wild type model, Y183H and Y183F models. The two-sequences alignments were obtained using LALIGN [In order to model the 3D structure of the involved proteins, we used Modeller 9.24   and luciferase activity in the cellular lysates was assessed on a Glomax 20/20 luminometer (Promega) using the Luciferase Assay System (Promega). Results were normalized for transfection efficiency by using a GFP coding vector (1 μg). All luciferase results represent the average ±S.D. of three independent experiments. All samples were read in triplicate.t-test (GraphPad Prism4 software) .Protein amounts were quantified by densitometry using the ImageJ software. Experiments were performed at least in triplicate and error bars represent standard error of the mean (S.E.M.). Data were statistically analyzed using Student’s We have previously discovered the interaction between RAB7A and vimentin by a two-hybrid screening and confirmed it with different techniques, including co-immunoprecipitation in HeLa cells . HoweverLysates of HeLa cells expressing HA-tagged RAB7A wild type and myc-tagged wild type or mutated vimentin were subjected to co-immunoprecipitation using anti-HA monoclonal antibody covalently attached to cross-linked agarose beads. Samples were run on SDS-PAGE and subjected to Western blot analysis. Interestingly, we found that RAB7A interacts with vimentin 1-256 and vimentin 92-256 (containing coil1 domain). As the coil1 domain is composed of the coil1A and coil1B domains, we evaluated the interaction of vimentin 1-141 (encoding head and coil1A domains) and vimentin 142-256 (encoding coil 1B) with RAB7A, demonstrating that RAB7A also interacts with these fragments but it does not interact with vimentin 256-411 and vimentin 256-466 (containing coil2 domain) fragments b. These To analyze structurally this interaction, we performed docking simulations using Gramm-X (for the whole model) or PatchDock (for the fragments), as detailed in Materials and Methods. This model confirmed the co-immunoprecipitation data as it revealed the interaction of RAB7A with the vimentin coil 1B domain but also with the vimentin head domain, while no interaction was detected with coil2 or tail domains .Interestingly, a homology search revealed that a string of about fifty amino acid in the coil1B domain of vimentin displays 30% of identity and 52% similarity with the RAB7A-interacting domain of Rab interacting lysosomal protein (RILP), described previously . MoreoveThus, we demonstrated that vimentin contains at least two domains interacting with RAB7A and one of them shares similarity with the RAB7A-interacting domain of RILP.Y183H and RAB7AY183F mutants discovering that they show a higher energy value for both Y183H and Y183F models (an increase of 15% for RAB7AY183H and 13% for RAB7AY183F) if compared with the wild-type protein . Finally, for both RAB7AY183H and RAB7AY183F a flexibility increase was observed in the 127 to 134 region and at the C-terminal domain of RAB7A, while an opposite effect was shown in the domains around residues 92 and 36.To test if RAB7A phosphorylation is required for the interaction with vimentin, we performed a co-immunoprecipitation assay in HeLa cells expressing myc-tagged vimentin and HA-tagged RAB7A wild type, Y183H or Y183F. Interestingly, both RAB7A mutants were not able to interact with vimentin suggesting that dephosphorylated RAB7A cannot interact with its effector d. As shoFireDock e left deFireDock . Finally RAB7AWT . In partGiven the regulation of RAB7A on vimentin phosphorylation state, we have analyzed the activation state of AKT, PKA, PAK1, and ROCK2 upon RAB7A silencing in HeLa cells by evaluating the abundance of these kinases and of their activated forms through Western blot analysis a.We observed that depletion of RAB7A did not alter PKA and ROCK2 abundance or activity a. HoweveHaving demonstrated that RAB7A depletion influences AKT and PAK1 kinases and given the fact that over-expression of RAB7A determined an increase in the amount of Ser38 and Ser55 phosphorylated vimentin , we analTo further validate our data, we treated HeLa cells with another RAB7A siRNA and we evaluated PAK1 and AKT abundance and activation. As expected, using both siRNA, we obtained similar results c. IndeedY183F, to test if the expression of RAB7A mutant protein also diminished AKT activation and phosphorylated PAK1. Interestingly, similarly to RAB7 silencing, the expression of RAB7AY183F reduced P-AKT (Phosphorylated-AKT) and P-PAK1 (Phosphorylated-PAK1) abundance in HeLa cells  by several kinases and this modification leads to increased transactivation . Among t Ras V12 e.Altogether these data indicate that RAB7A affects NF-kB expression and its nuclear localization while it does not influence NF-kB activation.It was previously demonstrated that RAB7A has a role in cell migration . CofilinY183F expression on cofilin-1 abundance, we transfected HeLa cells with plasmids coding for HA or HA-RAB7AY183F mutant protein and, through Western blot analysis, we demonstrated that the mutant protein diminished cofilin-1 abundance  or EGF stimulation assays  and in NRecently, it has been proved that regulation of vimentin phosphorylation and assembly are important for the interaction between vimentin and β1-integrin and for cell adhesiveness on fibronectin substrate . Indeed,AKT and PAK kinases are also key regulators of proliferation, apoptosis and survival and they are overexpressed or hyperactivated in different types of cancers ,42. ModuY183F mutant protein was not able to induce the translocation of NF-kB in the nucleus. These results are in line with evidence from the literature showing that NF-kB-dependent transcription is not regulated only by p65 nuclear localization, suggesting the existence of other factors required for NF-kB activation [We also evaluated the effects on NF-kB, a transcription factor constitutively active in most cancers, controlling proliferation, angiogenesis, invasion, metastasis, chemoresistance, and radioresistance . Normalltivation . Accorditivation . Thus, RThe rates of polymerization and depolymerization of actin cytoskeleton are determinant for cell motility and cofilin-1 is fundamental for actin dynamics ,68. We fY183F is not able to rescue RAB7A activity on PAK1 and AKT after silencing and its expression does not cause nuclear translocation of NF-kB. The possible involvement of phosphorylation of this residue in these events needs further investigation.Importantly, we found that mutating the RAB7A Y183 amino acid changes the structure of the protein affecting the interaction with vimentin and, consequently, mutant proteins are not able to act as the wild-type. Indeed, for instance, RAB7ANotably, we found that RAB7A depletion affects MMP2 activity but not abundance of pro and active MMP2 in the whole cell lysates. A possible explanation of these results could be that in Rab7-depleted cells there is a defective secretion of MMP2. This has been previously reported in cortactin silenced cells, where MMP2 abundance in cell lysates was not affected while abundance and activity in conditioned medium were decreased, suggesting that cortactin is important for MMPs secretion . ImportaThe link between RAB7A and MMPs could be relevant for potential clinical applications. The use of MMP inhibitors in clinical trials was unsuccessful possibly because of the lack of specificity of these drugs towards MMPs . FailureIn conclusion, we proved that RAB7A influences AKT and PAK activity, regulating, also, several of their downstream effectors and impacting on the reorganization of vimentin filaments, which favor cell migration .


Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and–H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system.SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in  Drosophila mutant strains, sf3b1-H698D and -H698R using CRISPR-Cas9. Analyses of these two fly strains find that the two oppositely charged Sf3b1-H698 mutants are defective in development. In addition, one mutant has decreased climbing ability, whereas the other mutant has impaired immune response. Further RNA-seq allows us to find responsible genes in each mutant strain, and lariat sequencing reveals that both mutations cause aberrant selection of the intronic branch sites. Our findings provide the first in vivo evidence that Sf3b1 mutations result in defective development, and also reveal a molecular mechanism of these hotspot histidine mutations that enhance the use of cryptic branch sites to alter splicing. Importantly, we demonstrate that the H698R mutant prefers to use far upstream branch sites.In the past decade, one of the important findings in the RNA splicing field has been that somatic SF3B1 mutations widely occur in many cancers. Including R625, H662, K666, K700 and E902, there are five hotspot mutation sites in the highly conserved HEAT repeats of SF3B1. Several kinds of H662 mutations have been found widely in MDS, AML, CLL and breast cancers; however, it remains unclear how these H662 mutations alter splicing and whether they have in vivo effects on development. To address these questions, in this manuscript, we first summarized the H662 mutations in human diseases and constructed two corresponding  Studiesact . As the act ,34–37. Sact ,38,39, Sact  and Prp3act .Most of the SF3B1 mutations are located in its HEAT repeats, especially in HEATs 4–12, where five residues, R625, H662, K666, K700 and E902, are hotspots and most of them exhibit cancer lineage specificities ,42,43. MTranscriptome analyses of human cell lines and mouse models have found that alternative 3′ splice site (A3SS) events are enriched in SF3B1 mutation-mediated splicing changes, in which upstream cryptic 3′SSs are preferentially used ,47,57–59Sf3b1 mutations have also been studied for genetic interactions and for the sensitization of clinically relevant drugs, as well as the developmental and splicing defects in C. elegans [Sf3b1 mutants have revealed that Sf3b1 is essential for the neural crest development [sf3b1 mutants in Drosophila have not been previously investigated. elegans . In zebrelopment  and the elopment , and regelopment . Howeversf3b1-H698D and -H698R, in which the homologous residue His698 in Drosophila Sf3b1 was mutated to Aspartic acid (D) and Arginine (R), respectively. Both mutants exhibit defects in development; the H698D mutant has an impaired immune response to fungal infection, whereas the H698R mutant has a decreased ability in climbing. RNA-seq allowed us to find responsible candidate genes in each mutant, and lariat sequencing revealed that both mutations cause aberrant selection of branch sites and that the H698R mutant prefers to use upstream branch sites.Several H662 mutations, including H662D and H662R, two oppositely-charged residues, have been found in multiple cancers without obvious type preference as mentioned above. However, it remains unclear how these H662 mutations alter splicing and whether they have in vivo effects on development. To test these mutations in an in vivo model system, we constructed two mutant fly strains, Drosophila strains with mutations at H698, the corresponding residue of human H662, using the CRISPR-Cas9 system. Since our previous data in yeast demonstrated that the D and R mutations of Hsh155/SF3B1 result in opposite splicing effects on branch site mutant reporters and opposite effects on interaction with Prp5 [sf3b1-H698D and -H698R, were constructed , survival was significantly impaired for H698D females and males, but not significantly changed for H698R females and only slightly decreased for H698R males , in which two lines of each sf3b1 mutant were sequenced for accuracy  family genes that are involved in the detoxification of foreign compounds, such as the tested Cyp6a-17 and Cyp12d, both of which are known to have monooxygenase activity and are induced by xenobiotic treatment [To validate this, we performed RT-qPCRs to test levels of the innate immune response genes, including in H698R . These dreatment .Using the common differential AS (DS) analysis tool rMATS, we identified 1,149 and 1,290 AS events that were significantly changed in the two mutants, respectively Figs . The mossf3b1 mutants (ΔPSI > 0.05), whereas no such enrichment was seen in inhibited RI events (ΔPSI < -0.05)   left. Se mutants  right. I mutants  middle. H698R flies , head and body from the WT adult  in the WT and sf3b1-H698 mutant backgrounds, respectively. Additional expression of the Mef2(RB) isoform doubled the climbing ability of the H698R mutant, while only ~15% increase of the climbing ability of the WT and H698D mutant  to evaluate all the individual SSs in the Drosophila transcriptome [H698D and H698R, respectively (rd nucleotide (C) of the 3′SS Differentectively . We furtectively ; whereas′SS Figs . These rsf3b1-H698 mutants on the 5′SSs preferentially results in intron retention and on the 3′SSs preferentially causes intron retention or alternative 3′SS.We then compared the two data sets of ΔUSS and ΔPSI, and found that the significantly usage-changed 5′SSs were highly enriched only in the group of RI events  left, whHsh155-H331D and–H331R using the well-established ACT1-CUP1 reporter system [Drosophila introns. To test this hypothesis, we sequenced nested PCR products of the reverse transcribed lariats from four A3SS events to identify BSs used in the WT and sf3b1 mutants  the mutated histidine residue may have a different influence on the conformational changes of Sf3b1 during the transition between spliceosomal complexes compared to other HEAT motif mutations. For example, many identified intron retention events in this study are connected with the decreased usage of 5′SSs, and those introns are relatively short, a characteristic of fly introns in comparison to mammalian introns.Differential A3SS events have been found to be the dominant splice changes in other studied SF3B1 mutations, such as K700E, K666 mutations and R625 mutations in human cells lines ,47,59 an mutants . This coHsh155-H331D mutation decreases yeast Hsh155/SF3B1 interaction with Prp5, whereas the Hsh155-H331R mutation enhances interaction with Prp5, and they have opposite splicing effects on suboptimal BS region substrates, indicating that selection specificities of the branch sites by these two mutants are different. In this study, we found that there are ~1,000 changed AS events in each sf3b1-H698 mutant fly. Sequencing of lariat products from four changed A3SS events, we demonstrate that aberrant branch sites are used in the two sf3b1-H698 mutants, of which the H698R mutant prefers to use far upstream cryptic branch sites, showing a different characteristic from the H698D mutant. The human H662 residue of SF3B1 directly interacts with the intronic 13th-15th nucleotides upstream of the branch site adenosine in the Cryo-EM structure of Bact complex [Drosophila His698 residue mutation to a stronger positively-charged Arg residue results in either a disordered open-state conformation of Sf3b1 that is defective in Sf3b1—Prp5 interaction, or a less stable close-state conformation that alters the pre-mRNA—Sf3b1 interaction  Drosophila melanogaster used in this study is a w1118 isogenic strain (BDSC 5905). Point mutant strains were constructed using the CRISPR/Cas9 system [nanos-Cas9 by UniHuaii Technology Company. Specific primers that distinguished point mutations were used for genomic PCRs to screen for the desired alleles, which were further validated by Sanger sequencing of amplicons. The flies obtained were then crossed for at least five generations with the WT strain to eliminate potential off-target events. Homozygous point mutant flies were maintained and cultured on standard cornmeal agar medium. All primers and oligos used are listed in The wild type  strain was constructed through incorporating pUAS-T(CDS of Mef2-RB) at attP2 site on the 3rd Chr. Then sf3b1-H698 mutant strains were separately crossed with Mef2-GAL4 and UAS-Mef2(RB), and the strains of sf3b1-H698R/H698R;Mef2-GAL4/UAS-Mef2(RB) and sf3b1-H698D/H698D;Mef2-GAL4/UAS-Mef2(RB) were finally obtained by further crossing.Western blot signals of Sf3b1 and Tubulin were detected using Rabbit anti-Sf3b1 antibody (antigen: VDEDEDDGFPVPQKRT) and anti-tubulin antibody (Sigma), respectively. Fat bodies of third-instar larvae were dissected, followed by incubation with the primary antibody Rabbit anti-Sf3b1 (1:500) and then the secondary antibody goat anti-rabbit Alexa Fluor 594. DAPI  was used for staining the nuclei. Images were acquired using a Carl Zeiss LSM880 confocal microscope.The number of eggs laid per female fly was measured as described . BrieflyHomozygous flies were mated and their laid eggs were collected in a 0.5_hr window and counted as time zero. The 1st instar larvae (270 for each strain) were picked and transferred into new vials with standard food at the 30_hrs post-laying. Pupation and eclosion of flies were counted in regular intervals . Their lDrosophila were dissected and obtained as described [Climbing ability (negative geotaxis) was measured as described . Ten fliescribed .7 spores/ml of Metarhizium anisopliae ARSEF 23 (Ma23). Briefly, after gentle shaking to evenly distribute the spores after bathing, 150 flies per sample were moved into fresh vials with food. Non-infection controls were given the same treatment without fungus. Flies were then kept at 29°C and transferred to new vials every two days with counting of the surviving flies. Percentages of survivals were presented by the averages with standard errors, and the median survival days were calculated using GraphPad Prism [Fungal infection was carried out with 10ad Prism .Drosophila melanogaster genome (dm6) by Hisat [Total RNAs from the 5d_adults were isolated by TRIzol (Ambion) and treated with RNase-free DNase I (Invitrogen). Construction of cDNA libraries and sequencing were performed using Illumina HiseqXten-PE150 by Novogene. Raw reads from RNA-seq were quality filtered and trimmed, and then mapped to the by Hisat  and counby Hisat .Analysis of differentially expressed genes was performed by DEseq2, genes with fold changes > 1.5 and FDR < 0.05 in both two lines were screened as significant. The Gene Ontology (GO) enrichment is analyzed by online DAVID. Differentially spliced (DS) events were analyzed by rMATS . SignifiΔΔCt analysis.For the regular RT-PCR, reverse transcription was performed using RevertAid Reverse Transcriptase (Thermo), and the cDNA was amplified by Ex-Taq (TaKaRa). qPCR was carried out using SYBR Green Master Mix (Applied Biosystems) in biological triplicates followed by The muscle samples for RT-PCR were dissected as described  with modst-step PCR was performed using primers P1′ and P1 in 25 μl for 30 cycles, and the 2nd-step PCR was carried out with primers P2′ and P2 in 50 μl after adding 0.2 μl product from the 1st-step for 35 cycles. One-fourth (12.5 μl) of the PCR products were fully separated by 4% agarose for visualization of bands; the remaining PCR products were running on 4% agarose for short time, gel purified by an Axygen kit and then subcloned into a T-vector (Takara). To identify branch sites and their usage frequencies, 10–12 subclones from each strain were picked for Sanger sequencing, and their sequences were aligned according to the junctions between 5′SSs and BSs. All the primers used are listed in For the lariat RT-PCR, cDNA was synthesized by SuperScript IV Reverse Transcriptase (Invitrogen) using an intron-specific antisense primer P1′  The mutant strains were screened and validated by genomic PCR and sequencing. Allele specific primers were used for the WT, H698D and H698R strains respectively, while common primers were used for Sanger sequencing. (B) mRNA and protein levels of Sf3b1 were not considerably changed in the two mutant strains. Left, RT-PCR for detection of sf3b1 mRNAs; right, western blot for detection of Sf3b1 protein. (C) Cellular location of Sf3b1 mutations is similar to the WT protein. Cells from the fat body were used for immunohistochemistry. DAPI (blue) defines the region of the nucleus, Sf3b1 was visualized by Alex-594 (red). Images in panels B and C are representatives from multiple assays.((TIF)Click here for additional data file.S2 FigA) Fewer eggs were laid in the early stage by sf3b1-H689D and -H698R mutants. The laid-eggs were counted from females crossed with males from their own strains. (B) Decreased egg-laying of sf3b1 mutants. In comparison to the WT, females of the two sf3b1 mutants laid significantly fewer eggs during the first half of test time. Means: WT = 274.1, H698D = 186.1, p = 0.0015, H698R = 182.3, p = 0.0002. Females of the two sf3b1 mutants laid no significantly changed eggs during the late half of test time. Means: WT = 175, H698D = 195.5, p = 0.5296, H698R = 173.4, p = 0.9437. Data represent the mean ± SEM from ten samples from each strain. (C) Shorter lifespans of the sf3b1 mutants. Median survival days were measured and listed. Median survival: WT = 72, D = 58, p < 0.0001 (****), R = 58, p < 0.0001 (****).((TIF)Click here for additional data file.S3 FigA) Correlation analysis of overall transcriptomic genes expression between the two lines of each sf3b1 mutants. (B) Correlation analysis of differentially expressed genes’ log2FoldChange between the two lines of each mutant.((TIF)Click here for additional data file.S4 FigFor each mutant, five types of events are shown. Bar in light colors, ΔPSI decreased events; bar in dark colors, ΔPSI increased events.(TIF)Click here for additional data file.S5 FigAS events were randomly picked from results of the rMATs analysis, and RT-PCR were performed multiple times from biological samples.(TIF)Click here for additional data file.S6 FigH698D; green dots: events in H698R; black dots with gene labeling: unique events in H698R.Orange dots, events in (TIF)Click here for additional data file.S7 FigWT strain are shown in red, sf3b1-H698D in blue, and sf3b1-H698R in green. Numbers of all the exon-exon junction reads are indicated, and the AS changed exons or introns are shown in red at the bottom of each panel.Data of the (TIF)Click here for additional data file.S8 FigMef2 are obtained from Flybase and confirmed by our RNA-seq data. The isoforms are named by Flybase and we number the exons considering both clarity and consistency. Blue rectangles: CDS exons, blue boxes: UTRs, and number of amino acids from each isoform-coded protein, if expressed, are also listed. Major changed isoforms in the sf3b1-H698R strain are indicated.Eleven isoforms of (TIF)Click here for additional data file.S9 FigH698D and H698R and compared with the usage-not-changed SSs (|ΔUSS| < 0.01). Blue: ΔUSS > 0.01, brown: ΔUSS < -0.01, grey: |ΔUSS| < 0.01. Values of mean, median, and SE from each group are presented.The 5′ and 3′SSs with |ΔUSS| > 0.01, FDR < 0.05 were screened in (TIF)Click here for additional data file.S10 FigRilpl-intron 2 (A), bip2-intron 5 (B), bol-intron 7 (C) and Mef2-intron 10 (D) were sequenced and aligned, and the usage frequencies of BSs were calculated for each strain.PCR products from the amplified lariats of (TIF)Click here for additional data file.S11 FigA) SF3B1 interacts with the BS—U2 snRNA duplex. (B) A zoom on the region around the H662 residue that interacts with the upstream of BS. Cyan, SF3B1; orange, pre-mRNA; violet, U2 snRNA; yellow sticks, H662 residue; red, BS-A (branch site adenosine); numbers, nucleotide positions of the pre-mRNA from the BS to upstream. This figure is rendered from PDB ID: 5Z56  using PyMOL.((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Escherichia coli (STEC) O26 is the predominant non-O157 serogroup causing hemolytic uremic syndrome worldwide. Moreover, the serogroup is highly dynamic and harbors several pathogenic clones. Here, we investigated the phylogenetic relationship of STEC O26 at a global level based on 1,367 strains from 20 countries deposited in NCBI and Enterobase databases. The whole-genome-based analysis identified a new genetic clade, called ST29C4. The new clade was unique in terms of multilocus sequence type (ST29), CRISPR (group Ia), and dominant plasmid gene profile (ehxA+/katP-/espP-/etpD-). Moreover, the combination of multiple typing methods  demonstrated that this new lineage ST29C4 was in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains. Besides, we observed that ST29C4 harbored extraintestinal pathogenic E. coli (ExPEC)-related virulence gene (VG), tsh, and STEC-associated VG, stx2a, suggesting the emergence of a hybrid pathogen. The ST29C4 strains also exhibited high similarity in stx2a-prophage and integrase with the O104:H4 strain, further demonstrating its potential risk to human health. Collectively, the large-scale phylogenetic analysis extends the understanding of the clonal structure of O26 strains and provides new insights for O26 strain microevolution.Shiga toxin-producing IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) O26 is the second prevalent STEC serogroup only to O157, which can cause a series of diseases ranging from mild diarrhea to life-threatening hemolytic uremic syndrome (HUS). The serogroup is highly diverse and multiple clones are characterized, including ST29C1-C3 and ST21C1-C2. However, the phylogenetic relationship of these clones remains fully unclear. In this study, we revealed a new genetic clade among O26 strains, ST29C4, which was unique in terms of CRISPR, multilocus sequence type (MLST), and plasmid gene profile (PGP). Moreover, the combination of multiple typing methods demonstrated that this new clone was located in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains . Overall, the large-scale phylogenetic analysis extends our current understanding of O26 microevolution. Escherichia coli (STEC) is recognized as the important zoonotic pathogen transmitted to humans through food, water, and animals  plasmid gene profiles (PGP) that consist of four plasmid-borne genes ; and (iv) clustered regularly interspaced short palindromic repeats (CRISPR) spacer contents. Before the 1990s, most O26:H11 strains isolated from clinical samples possessed stx1a alone or rarely together with stx2a (etpD)  . Moreovea (etpD) .stx2a alone was reported in Germany (katP and espP). Subsequently, a distinct ST29 clone (referred to as the “new French clone”) that was negative for all the PGP-related genes (ehxA-/katP-/espP-/etpD-) emerged in France .Since the 1990s, the O26 strains belonging to another MLST type, ST29, have been frequently detected from clinical patients , 19. In  Germany . Since t Germany . This nen France . This clhia coli . Moreovein Japan . The JapThe epidemic of these various O26 clones around the world has demonstrated the genetic diversity of STEC O26. So far, several studies have started to investigate the clonal structure of O26:H11 strains , 17. Howin silico whole-genome sequence (WGS) quality control analysis demonstrated that all 1,367 strains used in this study were positive for the O26wzx or O26wzy genes and harbored the fliC H11 allele. These strains were distributed in 20 countries (n = 386), Japan (n = 328), USA (n = 275), New Zealand (n = 111), or Australia (n =53)  or ST29 , and a small number of isolates were the variants of ST21  or ST29   . Of them, 0.66%) .To present a global phylogenetic overview for O26 strains, the ML phylogenetic tree based on 35,668 recombination-free SNP sites on a 3,037,060 bp core genome sequence was constructed. Apart from previously described five lineages (ST29C1-C3 and ST21C1-C2) based on PGP and SNP differences , the larTo test whether the evolutionary divergence of O26:H11 is reflected by CRISPR, a minimum spanning tree based on the presence or absence of CRISPR spacer was constructed. As shown in To further explore the divergence between and within O26 lineages in spacer contents, all O26 strains were subtyped based on the organization of CRISPR spacer. The spacer arrangements of CRISPR1 and CRISPR2a loci in all O26 strains were summarized in When further analyzing the spacer difference between groups, all the alleles in group I (ST29C3-C4) differed from those in group II  by the spacers at position 4 to 7 and 12 to 13 in CRISPR2a . The tracas genes  for 1334 strains were concatenated to create a phylogenetic tree to further evaluate the phylogenetic relationship of all O26 lineages. Similar to the result of CRISPR clustering, the phylogenetic tree based on a complete cas gene cluster supported the middle location of ST29C4 between ST29C3 and other lineages .Accumulating evidence suggested CRISPR could coevolve with associated as genes . Thus, aehxA+/katP-/espP-/etpD with a positive rate of 75% (12/16). Moreover, this predominant PGP in ST29C4 was occasionally detected in ST21C1 (1 of 657 strains), ST29C1 (1 of 66 strains), or ST29C2 (1 of 61 strains). The second most prevalent PGP in ST29C4 was ehxA+/katP+/espP-/etpD with a positive rate of 18.75% (3/16), which was identified in 10 ST21C1 strains. The least frequent PGP (ehxA-/katP-/espP-/etpD-), which was previously recognized as a key characteristic of the new French clone   was detected in ST21C1, ST21C2, and ST29C2 strains, whereas the other stx1a gene  was specifically detected in ST21C1 strains. As shown in stx2a genes also showed an uneven distribution among O26 lineages except that the stx2a_2 gene was detected in all O26 lineages. A BLASTn search of the stx2a_2 gene demonstrated that it was identical to that of the O104:H4 strain  that caused the 2011 STEC epidemic in Europe. Notably, the stx2a gene sequences in ST29C4 strains were also the same as that of O104:H4 strains.We next characterized the 2a alone . Uniquel strains . The firstx2a-harboring prophages in 6 ST29C4 strains and O104:H4 strain  were compared. As shown in wrbA gene. The remaining stx2a-prophage regions in the other 4 strains also showed high similarity to that of the O104:H4 strain, although their integrase genes and insertion sites of stx2a-prophage in the other 4 strains could be not determined due to truncated genome sequences.Subsequently, the eae, iha, lpfAO26, efa1, paa), type III secretion system effector , type II secretion system , aerobactin , yersiniabactin  and temperature-sensitive hemagglutinin (tsh).To further reveal the genetic difference among O26 lineages, we analyzed the prevalence of various virulence genes (VGs). A total of 67 VGs was detected in at least one O26 strain. These VGs detected included genes encoding adhesins , was identified in all strains but five strains -Ib, APH(6)-Id, sul2, and tet(C).We next detected the resistance genes related to 11 classes of antibiotics among all O26 lineages. A total of 531 O26 strains  contained at least one antibiotic resistance gene. Also, there was a difference in the numbers of acquired ARGs between O26 lineages. The mean numbers of acquired ARGs were 1.93 in ST21C1, 1.14 in ST21C2, 1.52 in ST29C1, 0.59 in ST29C2, 1.35 in ST29C3, and 0.25 in ST29C4. For ST29C4, only one strain carried resistance genes, including STEC O26:H11/H− has emerged as the main causative pathogen of HUS, second only to STEC O157 . The WGScas genes   encodes enterohemolysin (ehxA), catalase-peroxidase (katp), serine protease (espP), and type II effector (etpD), has been identified in most STEC O26 strains . The other three PGP-related genes  were absent in most ST29C4 strains but present in most ST29C1-C2 and ST21C1-C2 strains  . The com strains . Lineage strains , 33. How strains . This instx genes  and stx2 (stx2a to stx2k) (E. coli (EAEC) O104:H4 caused one of the largest foodborne outbreaks in Germany in 2011 through the acquisition of stx2a-harboring phage  encoded by tx genes . The stxo stx2k) . It is png phage . The acq strains . In thistsh gene. Nevertheless, the tsh gene was not found in any strain from other lineages, suggesting an independent acquisition event. The tsh gene encodes a temperature-sensitive hemagglutinin and is often located on the ColV virulence plasmid in avian pathogenic E. coli (APEC)  (stx2a and tsh genes in six ST29C4 strains was identified (Table S1). The combination of STEC virulence determinants and other pathogroup-defining virulence genes is not rare, contributing to the emergence of hybrid pathogens. Many investigations have elaborated on the prevalence and clinical significance of hybrid E. coli, such as STEC/ETEC, STEC/EAEC, and STEC/UPEC hybrids (Another unexpected finding of ST29C4 strains was that 11 ST29C4 strains possessed the i (APEC) . Besides (ExPEC) . Here, t hybrids , 39, 40.Salmonella typhimurium showed that the discriminatory power of genotyping based on the combination of CRISPR, and other molecular markers were comparable with pulsed-field gel electrophoresis (PFGE)  . The higs (PFGE) –31, 43.  (PFGE) –. The com (PFGE) –, 44. Sar (PFGE) – reportedThe WGS-based phylogenetic analysis identified a new genetic clade, designated ST29C4, which displayed genetic uniqueness in terms of MLST, PGP, and CRISPR. Moreover, the combination of multiple typing methods demonstrated that ST29C4 was in the intermediated phylogenetic position between ST29C3 and other lineages . Overall, these findings extend our current understanding of O26 strain microevolution.Escherichia coli O26 isolates were retrieved from the National Center for Biotechnology Information  and EnteroBase databases (http://enterobase.warwick.ac.uk/species/ecoli) (retrieved in November 2020). The read or assembled sequence data of O26:H11 strains were downloaded after confirming the serotyping using the EcOH database. Meanwhile, low-quality sequences were excluded . In some cases, the genome sequence of the same isolate was recorded repeatedly in the two databases. Such duplicates were removed by identifying the biosample accession number. Besides, duplicated strains that harbored identical recombination-free core genome alignment were also removed to construct a nonredundant database . Finally, a total of 1,367 O26:H11 strains collected worldwide were included in this study. Specific metadata for each isolate  was collected from public databases (Table S1). Among them, 28 isolates lacked at least one of the three above-mentioned metadata. The missing metadata was regarded as unknown. The host sources of isolates were categorized into six groups: human, bovine, other animals, food, environment, and unknown source (n source . The rawAP010953.1) was used as a reference genome for phylogenetic analysis. PHASTER and ISfinder were used to identify the prophage and IS (insertion sequence) region located on the reference genome, and then a phage- and IS-masked reference genome was obtained using Bedtools v2.30 (https://github.com/tseemann/snippy) and the alignment result was delivered to gubbins v3.0.0  analysis was performed to further infer the phylogenetic relationship of all O26 strains. An E. coli cgMLST scheme comprising 2,513 target loci of E. coli was employed to acquire the cgMLST type based on genomic sequences  using the seven housekeeping genes  as queries (https://cge.cbs.dtu.dk/services/MLST/). The reference nucleotide sequences of stx1, stx2, and eae variants were publicly available and downloaded from GenBank. The stx and eae genotyping of each strain were determined by in silico BLASTn search using stx and eae reference sequences as queries, respectively (identity ≥ 99% and coverage ≥ 99%)  related to PGP analysis was determined by VirulenceFinder 2.0 on the CGE webserver (identity ≥ 99% and coverage ≥ 99%) (equences . The mine ≥ 99%) . The pree ≥ 99%) .The CRISPR recognition tool was used for identifying CRISPR arrays and extracting spacer sequences . The setcas gene cluster was present in O26 strains  as the reference sequence (identity ≥ 85% and coverage ≥ 95%). Type I-E cas gene clusters were annotated in strain 11,368 . Once the complete cas gene cluster for a strain was determined, all cas genes for the strain were concatenated to create an ML tree to estimate the phylogenetic relationship of strains.A previous investigation suggested that one typical type I-E  strains . The seastx-prophage was implemented through the screening of the stx gene in all predicated prophages using BLASTn search. The comparison of stx-prophage was performed by EasyFig  (identity ≥ 95% and coverage ≥ 95%). The pipeline contained known VFs and acquired ARGs for E. coli, which were taken from the VFDB (http://www.mgc.ac.cn/VFs/main.htm) and CARD databases (https://card.mcmaster.ca/).The presence of other virulence factors and acquired ARGs associated with


Tumor heterogeneity includes variable and fluctuating oxygen concentrations, which result in the accumulation of hypoxic regions in most solid tumors. Tumor hypoxia leads to increased therapy resistance and has been linked to genomic instability. Here, we tested the hypothesis that exposure to levels of hypoxia that cause replication stress could increase APOBEC activity and the accumulation of APOBEC-mediated mutations. APOBEC-dependent mutational signatures have been well-characterized, although the physiological conditions which underpin them have not been described. We demonstrate that fluctuating/cyclic hypoxic conditions which lead to replication catastrophe induce the expression and activity of APOBEC3B. In contrast, stable/chronic hypoxic conditions which induce replication stress in the absence of DNA damage are not sufficient to induce APOBEC3B. Most importantly, the number of APOBEC-mediated mutations in patient tumors correlated with a hypoxia signature. Together, our data support the conclusion that hypoxia-induced replication catastrophe drives genomic instability in tumors, specifically through increasing the activity of APOBEC3B. Hypoxia, which is defined as conditions of insufficient oxygen, is associated with the resistance of tumors to standard therapies and poor patient prognosis ,2. Tumor2) most associated with therapy resistance, referred to as radiobiological hypoxia, lead to replication stress  proteins are a family of conserved cytidine deaminases that play a critical role in viral immunity ,14. APOBHIF1a+/+ and RKOHIF1a−/− (2. All cell lines were verified mycoplasma free using a HEK-Blue™ detection kit (Invivogen). Inhibitors/drugs used were: VX-970 (M6620) (MedChemExpress), AZD6738 (MedChemExpress), Gö6976 (Sigma Aldrich), MK-8776 (Selleckchem), Bay11-7085 (Tocris Biosciences), PHA-767491 (Selleckchem), N’ acetylcysteine (NAC) (Sigma Aldrich), and roscovitine (Ros) (Selleckchem). For siRNA-mediated knockdowns, RKO cells were transfected with siRNA to a final concentration of 50 nM using DharmaFECT 1  following manufacturer's protocols. Sequences of siRNAs used: siA3B (CCUGAUGGAUCCAGACACA[dT][dT]), sip53 (GUAAUCUACUGGGACGGAA[dT][dT]), and siATR (CAG GCA CTA ATT GTT CTT CAAd[T]d[T]).Colorectal RKO (ATCC), RKOHIF1a−/− , HCT116,2 were carried out in a Bactron II anaerobic chamber (Shel Labs) while all cyclic treatments were carried out in an M35 variable atmosphere workstation (Don Whitley Scientific). Oxygen concentrations were periodically validated using anaerobic oxygen indicator strips (Thermo Fisher) and an Oxylite oxygen probe (Oxford Optronix). Cells were seeded on glass dishes and harvested in the chamber with equilibrated solutions.Hypoxia treatments at <0.1% O2) were determined using the ΔΔct method using 18S as an endogenous control. Graphs were plotted using the mean of three biological replicates ± SEM. Primers sequences are given in Total RNA was isolated from cells with Trizol (Invitrogen/Life Technologies) and was reverse transcribed into cDNA with Verso enzyme kit (Thermo Scientific). SYBR Green PCR Master Mix kit (Applied Biosystems) was mixed with gene specific primers and the reaction was run on a StepOne Real-Time PCR System (Thermo Scientific). Fold change values relative to normoxia , 1X protease inhibitor (Roche) (added fresh), homogenized with a 25 G needle, and sonicated briefly in a Biorupter plus sonicator (Diagenode) before pre-clearing by centrifugation for 10 min. A deamination mix was made consisting of 8.25 ml of 5 μg/μl protein lysate, 0.625 μl RNaseA, 0.625 μl TE buffer, and 0.5 μl of 200 μM ssDNA oligo (5′-ATTATTATTANTCAAATGGATTTATTTATTTATTTATTTATTT’FAM3′) and incubated at 37°C for 3 h. Samples were then treated with 100 mM NaOH at 95°C for 30 min. Samples were mixed with Gel loading buffer II (Invitrogen) and incubated at 70°C for 3 min before being run on a 15% TBE-urea gel (Novex). Bands were analyzed with a Chemi-doc touch imaging system (Bio-Rad). Product bands were quantified using the analyze gels tool in ImageJ.Cells were lysed in HED lysis buffer and treated as with the deamination assay. Proteins were separated on a 4–20% polyacrylamide gel (Bio-Rad) and blotted onto a nitrocellulose membrane (Bio-Rad). Bands were imaged using Odyssey Infrared Imaging (LI-COR Biosciences). Experiments were carried out in triplicate and a representative blot is shown for each experiment. Antibodies used; Cell Signaling: p53-S15 (9284), RPA32 (2208), Chk1-S345 (2341), Chk1-S296 (90178), H3 (3638), H3-S10 (9701), p65 (3033), p65-S536 (93H1), cGAS (15102); Santa Cruz: p53 (sc-126), β-Actin (sc-69879), Chk1 (sc-8408), ATR (sc-515173); Bethyl: KAP1 (A300-274A), KAP1-S824 (A300-767A); Millipore: γH2AX (05–636), H2AX (07–627); Abcam: A3B (ab184990), Cyclin A (ab181591); BD Biosciences: HIF-1α (610958), GRP78 (610979).For BrdU incorporation plots, freshly made up BrdU (20 μM) was added in the dark to cells for 1 h before fixation in ice-cold 70% ethanol. Samples were treated with 2 M HCl and blocked in 2% FBS PBS solution. Propidium Iodide (Sigma Aldrich) was added to samples to determine total DNA content. Antibodies used were: BrdU (BD biosciences #347580), H3-S10  and Alexa Fluoro 488 (Invitrogen). Samples were run on a BD FACSCaliber machine (BD biosciences) and plots were analyzed with FloJo software.Cells were seeded onto autoclaved cover slips (Menzel-Glaser) before treatment. Cells were fixed with 4% fixation buffer (4% (w/v) paraformaldehyde in PBS). Samples were permeabilized with 1% PBS-Triton X-100 (Thermo Fisher) and blocked in 2% (w/v) BSA (Thermo Fisher) in 0.1% PBS-tween. For A3B staining, samples were permeabilized with 0.1% PBS–Triton X-100 (Thermo Fisher) and blocked in 5% (w/v) BSA (Thermo Fisher) in PBS. For EdU staining, cells were incubated with EdU (10 μM) (Thermo Fisher) and Click-iT Alexa Fluor 657 labelling kit (Thermo Fisher) was used for staining. Proteins were visualized with an LSM710 confocal microscope (Carl Zeiss Microscopy Ltd) and at least 150 cells were counted per condition. DNA damage and replication stress were quantified by counting cells with >5 53BP1 foci or >6 RPA foci respectively. Antibodies used: 53BP1 , A3B (Origene TA349029), RPA , RPA-S4/S8 (Abcam ab243866), Alexa Fluoro 488 (Invitrogen), and Alexa Fluoro 594 (Invitrogen).https://portal.gdc.cancer.gov/) to represent hypsig and p53genes respectively. The harmonized TCGA data were considered  was used for the comparison of more than two means. Statistical significance was assumed if P <0.05. Error bars represent mean ± standard error of the mean (SEM).A two-tailed, paired Student's 2) and changes to RPA and Chk1 were determined. Both RPA and Chk1 phosphorylation increased in hypoxia (<0.1% O2) but, as previously described, returned to near normoxic levels after longer exposure (20 hours)  is not involved in APOBEC expression as HIF1 is stabilized at both <0.1 and 2% O2. However, to confirm this, RKO cells with and without HIF1α were treated with hypoxia (<0.1% O2) and the levels of A3D and A3H determined. Induction of A3D in response to hypoxia was not changed by the lack of HIF1α while the induction of A3H in response to hypoxia increased further. These data demonstrate that neither A3D nor A3H were induced in a HIF1 dependent manner in hypoxia (<0.1% O2) (2) and hypoxic (<0.1% O2) conditions. Successful p53 knockdown was verified by the decrease in mRNA expression of INPP5D, a validated hypoxia-inducible p53 target gene (2)   was found between A3C, A3D, A3F, A3G, A3H and the mean score of the six hypoxia-induced p53-dependent genes in all these cancers . As predicted by our in vitro data, a negative correlation was seen between A3B and the mean expression of the 6 p53-dependent genes in all datasets, furthering the evidence for the decrease in A3B in hypoxia (<0.1% O2). However, as all members of the APOBEC3 family have deamination activity, we hypothesized that deamination could increase in response to replication stress in hypoxia as a result of increased expression of A3C, A3D, A3F and A3H, despite unchanged A3B levels. Using an in vitro deamination assay, we observed the expected increase in deamination in response to HU, however deamination was unchanged in both of the hypoxic conditions tested (2 and <0.1% O2)  . Importa2 Figure . Here, wpression  and the response  30,33).,33.2) anresponse . To veri0.1% O2) . Both A30.1% O2) . In supp0.1% O2) . Using sget gene  (30). As) Figure , F. Howe) Figure . To conf) Figure . Next, w) Figure . Previou) Figure ,35. Repr) Figure . Howevers Figure . The p53s Figure . At a si) Figure , L. Thes2) on DNA replication. RKO cells were exposed to HU or hypoxia and both propidium iodide (PI) and bromodeoxyuridine (BrdU) incorporation was analyzed  lead to a significant decrease in the mean fluorescence intensity (MFI) of the BrdU staining indicating decreased DNA replication. However, it was notable that the HU treated cells were incorporating significantly more BrdU compared to the hypoxic cells .To explain the contrasting biological responses to hypoxia and HU, we began with further investigation of the impact of both HU and hypoxia , which alone do not induce replication stress, might lead to increased replication and the DNA damage associated with replication catastrophe. Using a hypoxia chamber capable of rapidly switching between multiple oxygen concentrations, we investigated cyclic hypoxia conditions consisting of periods of <0.1% O2, which induce replication stress, and 2% O2 which leads to HIF stabilization but no replication stress  still led to replication stress by showing that Chk1 and RPA were phosphorylated in cyclic hypoxia , however these phosphorylations appeared to be maintained in cyclic hypoxia. To investigate further, we determined the number of cells with RPA foci in each condition and found that the percentage of cells with RPA foci increased over time in cyclic hypoxia, while in stable hypoxia they formed and decreased over time  leads to apoptosis this is not evident in cycling conditions . Fu. Fu2), wnditions . A G2 arnditions , I, J.2. Interestingly, when the cyclic hypoxic cells were released into 2% O2, a significant number of cells with 53BP1 and RPA foci remained 24 h later  and cyclic hypoxia. A robust increase in cGAS protein was seen in response to cyclic hypoxia while no increase was observed in stable hypoxia  or cyclic hypoxic conditions and western blotting was carried out for A3B. A 4-fold induction of A3B was seen in the cycling conditions but not stable hypoxia  lead to increased deamination suggesting that this is not dependent on the specific cyclic conditions used ..2) or cyTo investigate whether the increased A3B seen in cyclic hypoxia was a result of replication catastrophe, we began by co-staining for A3B and RPA. In response to both HU and cyclic hypoxia the nuclear intensity of A3B and the number of nuclear RPA foci increased. In contrast in stable hypoxia, only the number of cells with RPA foci increased Figure , B. Nextin vitro deamination assay  followed by western blotting for A3B. Surprisingly, inhibition of ATR abrogated the induction of A3B in cyclic hypoxia Figure . This way Figure , H. Thesy Figure . Howevery Figure . To furty Figure . Togethe2 lead to the induction of A3B expression and activity. Cyclic hypoxia is a physiologically relevant condition, although detailed study of this phenomenon has been hampered by difficulties in measuring the periodicity in patients. As of yet, there are no clinically useful markers of cyclic hypoxia in routine use  induces significant replication stress but this occurs in the apparent absence of DNA damage and does not lead to APOBEC expression/activity. In contrast, replication stress induced by either HU or cycling hypoxia leads to replication catastrophe and results in APOBEC expression/activity. The significance of our finding is highlighted by evidence suggesting that cycling hypoxia leads to increased APOBEC activity in patient samples.We have demonstrated that the biological response to replication stress is determined by the level of accompanying DNA damage (replication catastrophe). Exposure to hypoxia (<0.1% Oin vivo experiments suggest that cyclic hypoxia is commonplace in solid tumors (in vivo. The vast majority of studies utilize ambient air (21% O2) as the reoxygenation portion of their cyclic schedule . As APOBECs can also bind RNA as a substrate, mRNA editing has been proposed as a possible function of the family. Recently, a limited number of studies have implicated both A3A and A3G in mRNA editing (2)  . FurtherA number of previous reports have described increased replication stress signaling and a G2/M phase cell cycle arrest following A3B overexpression ,41. In cA limitation of our study is that A3A expression could not be detected in the RKO cell line which is significant as the generation of the APOBEC mutational signature in human cancers has been attributed to A3B and A3A . However2), cyclic hypoxia-induced p53 does not lead to apoptosis. Our findings demonstrating an increase in cell size and a G2 cell cycle arrest (Figures Further investigation is required to determine the fate of cells that experience replication catastrophe due to cyclic hypoxia. Interestingly, our data clearly demonstrate that in contrast to stable hypoxia (<0.1% O Figures , F, as w Figures . Howeverin vivo showed a significant A3B dependent effect on the speed at which tumors developed treatment resistance (in vitro (Targeting APOBECs is an emerging clinical strategy. Decreasing APOBEC expression could be used as an enhanced treatment strategy to limit tumorigenesis and the development of treatment resistance through a decreased mutation rate. Supportively, a study investigating the development of tamoxifen resistance sistance . Strategin vitro . The devin vitro . OverallAll data is available from the corresponding author upon request.gkab551_Supplemental_FilesClick here for additional data file.