An emerging new paradigm is that the organismal balance of these cations predominantly depends on a common gatekeeper, the channel-kinase TRPM7. Despite extensive electrophysiological studies and recent cryo-EM analysis, an open question is how the channel activity of TRPM7 is activated. Here, we performed site-directed mutagenesis of mouse TRPM7 in conjunction with patch-clamp assessment of whole-cell and single-channel activity and molecular dynamics (MD) simulations to show that the side chains of conserved N1097 form an inter-subunit Mg2+ regulatory site located in the lower channel gate of TRPM7. Our results suggest that intracellular Mg2+ binds to this site and stabilizes the TRPM7 channel in the closed state, whereas the removal of Mg2+ favours the opening of TRPM7. Hence, our study identifies the structural underpinnings through which the TRPM7 channel is controlled by cytosolic Mg2+, representing a new structure–function relationship not yet explored among TRPM channels.ZnThe online version contains supplementary material available at 10.1007/s00018-022-04192-7. We observed that the WT TRPM7 channel was inactive in the presence of both concentrations of free [Ba2+]i . The N1097Q variant showed significantly reduced currents only after administration of 1 mM free [Ba2+]i , whereas the N1098Q channel remained unaffected under both experimental conditions .Apart from Mgory site . Hence, 2+ and Mg·ATP inhibit TRPM7, most likely through different ligand binding sites i. The physiological intracellular concentrations of ATP vary between 2 and 9 mM in most mammalian cells i will recapitulate the effects of 6 mM [Mg·ATP]i co-applied with 250 μM free [Mg2+]i . We observed that the impact of the N1097Q and N1098Q mutations on the channel’s response to both Mg·nucletides were not different in such experimental settings .Previously, extensive electrophysiological analyses revealed that free Mgng sites , 13. Int channel , 13. Thean cells . In a phs ~50 μM  and it ws Mg·ATP . Using imentclasspt{minimai in the presence of the physiological range of [Mg2+]i concentrations using an internal solution containing 550 µM and 1 mM free [Mg2 +]i. We found that WT currents were entirely suppressed under these conditions . These results suggest that the N1097Q channel variant remained sensitive to Mg·ATP, but only in the presence of high concentrations of free Mg2+, in accord with the idea that Mg2+ and Mg·ATP independently interact with different TRPM7 channel sites i and [Mg·ATP]i or caused more general changes of regulatory characteristics of the channel, we studied the effects of small synthetic molecules acting as activators or inhibitors of the TRPM7 channel. First, we assessed the action of naltriben, a potent agonist of the TRPM7 channel [i and 550 µM free [Mg2+]i. The external application of naltriben led to a fast stimulation of WT and N1097Q currents  . The corresponding surrogate mean open times (TOS), an admittedly rough estimate for the duration of the open state, were 4.7 ± 0.6 ms and 1.5 ± 0.3 ms for the WT and N1097Q channel variants, respectively. Despite shortening of TOS, N1097Q did not affect the open probability (NPO) , suggesting that the N1097Q mutation facilitated gating motions in the TRPM7 protein. Interestingly, besides a 40 pS main level, at least one additional subconductance state for TRPM7 has been described i) pipette solution containing (in mM): 120 Cs·glutamate, 8 NaCl, 10 Cs-EGTA, 5 Cs-EDTA, 10 HEPES–CsOH, pH 7.2.Unless stated otherwise, a standard extracellular solution contained (in mM): 140 NaCl, 2.8 KCl, 1 CaCl2+]i and [Mg·ATP]i concentration–response data, the intracellular pipette solutions were prepared as outlined in Suppl. Tables S1–S4. Concentrations of [Mg·ATP]i and free [Mg2+]i were calculated using the Maxchelator software (maxchelator.stanford.edu).To obtain i-free intracellular solution and solutions containing 0.55 and 1 mM free [Ba2+]i (Suppl. Table S2). Middle panels: Representative I-V relationships obtained from individual ramps at 300 s in the Left panels. Right panels: Bar graphs of outward currents  obtained at 300 s as indicated in the Left panels. n, number of cells measured; n.s., not significant; **P< 0.01, *** P< 0.001 (ANOVA) (PDF 355 KB)Suppl. Figure S4. Inhibition of TRPM7 currents by cytosolic Mg·ATP and Mg·GTP. Whole-cell currents were measured in HEK293T cells transfected by WT , N1097Q  and N1098Q  variants of TRPM7 cDNAs (in pIRES2-EGFP). A, C, D Current amplitudes (mean ± SEM) were measured at +80 mV and plotted over time. Currents were measured using an intracellular solution containing 250 µM free [Mg2+]i without Mg·nucleotides, 250 µM free [Mg2+]i with 6 mM [Mg·ATP]i, and 250 µM free [Mg2+]i with 6 mM [Mg·GTP]i (Suppl. Table S3). B, D, F Bar graphs of outward currents  obtained at 300 s as indicated in . Note: The results obtained with 250 µM free [Mg2+]i without Mg·nucleotides and with 6 mM [Mg·ATP]i were taken from Fig. 3. n, number of cells measured; n.s., not significant; ***P< 0.001 (ANOVA) (PDF 216 KB)Suppl. Figure S5. Suppression of TRPM7 currents by 9 mM Mg·ATP in the presence of 1 mM free Mg2+. Whole-cell currents were measured and analysed analogously to the experiment outlined in Figure 3G, except that 9 mM [Mg·ATP]i and 1 mM free [Mg2+]i were included in the intracellular solution (Suppl. Table S4). n, number of cells measured; n.s., not significant; * P< 0.05, ** P< 0.01 (ANOVA). (PDF 61 KB)Suppl. Figure S6. Examination of TRPM7 currents in the perforated patch.Current-voltage (I-V) relationships of currents were measured in HEK293T cells transfected by WT (A), N1097Q (B) and N1098Q (C) variants of TRPM7 cDNAs. The I-V relationships were acquired after 25 s of breake-in using the standard intracellular solution containing 320 µM amphotericin B. D Bar graphs of outward currents  shown in (A–C). n, number of cells measured; **P< 0.01 (ANOVA) (PDF 49 KB)Suppl. Figure S7. Assessment of TRPM7 currents using a divalent cation-free (DVF) extracellular solution.Whole-cell currents of WT (A), N1097Q (B) and N1098Q (C) TRPM7 variants (in pIRES2-EGFP) expressed in HEK293T cells. Left panels: Current amplitudes (mean ± SEM) were measured at − 80 and + 80 mV and plotted over time. Currents were induced using the standard [Mg2+]i free intracellular solution and the standard external solution. When currents were fully activated, cells were perfused with the DVF solution as indicated by the black bars. Right panels: Representative I-V relationships obtained from individual ramps before (brown) and after (red) DVF application as indicated in the Left panels by coloured data points. D Bar graphs of outward  and inward  currents (mean ± SEM) shown in (A–C) at 200 s. n, number of cells measured; n.s., not significant (ANOVA). (PDF 230 KB)Suppl. Figure S8. Single-channel properties of the WT and N1097Q TRPM7 variants. Currents were recorded in outside-out membrane patches excised from HEK293 cells expressing WT  and N1097Q  variants of TRPM7. The intracellular solution was no-DIV (0 mM Mg2+). A, B Shown are current traces at a holding potential of − 60 mV from WT (A) and N1097Q channels (B), starting (from top to bottom) 2 s before channels were unblocked by switching from a divalent cation-containing bath solution (DIV) to a divalent-free bath (no-DIV) and 28 s and 238 s after solution exchange. C The left panel shows representative current traces from the same patches as in (A) and (B), obtained at the indicated patch potentials in a no-DIV bath. The right panel shows a plot of unitary current amplitudes versus patch potential constructed thereof, revealing a linear I-V relationships. The slope conductance (γs) derived from linear regression fitting (R2 = 0.997 and 0.997) was in these two outside-out patches 44 pS and 21 pS for WT and N1097Q channels, respectively. Insets: Statistical evaluation of outside-out recordings with the WT channel and N1097Q in the absence of intracellular Mg2+. Note that the open probability (NPO) was similar for the TRPM7 variants (P = 0.806), despite the significantly shorter open time (TOS) of N1097Q . The NPO and TOS data for N1097Q are taken from Fig. 7 and replotted here for comparison (PDF 524 KB)Suppl. Figure S9. Impact of intracellular Mg2+ and Mg·ATP on single-channel TRPM7 currents. Currents were recorded at a holding potential of – 60 mV in outside-out membrane patches excised from HEK293 cells expressing WT  and N1097Q (C–E) variants of TRPM7. The WT (A) or N1097Q (C) channels were induced by removing the extracellular divalent cations (DIV bath) using a no-DIV solution, as indicated above the current traces. The intracellular solution was no-DIV containing 3 mM Mg·ATP and 0.5 mM MgCl2, resulting in 2.9 mM [Mg·ATP]i and 250 μM free [Mg2+]i. Insets: currents on an expanded time scale from the segments indicated by arrows. The graphs on the right show all-point histograms from the two 30 s current traces. The dotted line indicates the closed level (C). The broken lines indicate the current level for 1 channel (O1), 2 channels (O2) or 3 channels (O3) being open. Single-channel amplitudes (i) taken from O1 were -2.4 pA and -1.4 pA for WT channels (A) and N1097Q (C), respectively. B, D The open probabilities (NPO) assessed for bins of 2 s over the whole 4.5 min duration of the experiments shown in . E Statistical evaluation of outside-out recordings with the N1097Q channel. The data obtained with 0 and 1 mM Mg2+ were replotted from Fig. 7. Note that intracellular Mg·ATP did not affect single-channel chord conductance , NPO  and open time  (PDF 175 KB)Suppl. Figure S10. Homology modelling of the S4-S5, S5, S6, and TRP domains of the open TRPM7 channel using the TRPV6 open channel as a template. A Sequence alignment of residues 982-1022 and 1071-1123 of TRPM7, and residues 471 to 513 and 553 to 605 of the human TRPV6 open channel (PDB 6BO8). B Overlap of the homology model (green) and the cryo-EM structure of the closed TRPM7  (PDF 97 KB)Suppl. Figure S11. The proposed arrangements of N1097 and N1098 in the lower channel gate of TRPM7. A Comparison of the interaction of Mg2+ at the lower gate in simulated WT and N1097Q channels. Protein residues that interact with Mg2+ are shown in yellow. Water molecules (WAT) that interact with the Mg2+ ion are shown as red and white spheres. B Representative snapshots of WT and N1098Q channels displaying hydrogen bond interactions (dashed lines) of residue 1098 (yellow) viewed intracellularly. N1097 (blue) and other residues that interact with the mutated residue in at least one subunit (white) are shown. Residues in diagonal subunit pairs are labelled in different colours. Note that the hydrogen bonds are transient, and the specific interactions shown are not present in all frames of the trajectory (PDF 208 KB)Supplementary file12 (DOCX 18 KB)Below is the link to the electronic supplementary material.


Extracellular-signal-regulated kinase (ERK) direct cell fate determination during the early development. The intricate interaction between the deposition of H3K9me2, de novo 5mC, and its oxides affects the remodeling of zygotic epigenetic modification. However, the role of fertilization-dependent ERK in the first cell cycle during zygotic reprogramming remains elusive.In the present study, we used the small molecule inhibitor to construct the rapid ERK1/2 inactivation system in early zygotes in mice. The pronuclear H3K9me2 deposition assay and the pre-implantation embryonic development ability were assessed to investigate the effect of fertilization-dependent ERK1/2 on zygotic reprogramming and developmental potential. Immunofluorescence and RT-PCR were performed to measure the 5mC or its oxides and H3K9me2 deposition, and the expression of related genes.We reported that zygotic ERK1/2 inhibition impaired the development competence of pre-implantation embryos. Following the ERK1/2 inhibition, H3K9me2, as well as 5mC and its oxides, were all accumulated abnormally, and the excess accumulation of paternal H3K9me2 and 5mC resulted in reduced asymmetry between parental pronuclei. Furthermore, ERK1/2 inhibition triggered paternal pronuclear localization of the H3K9 methyltransferase G9a and Tet methylcytosine dioxygenase 3 (Tet3). Moreover, the excess localization of G9a antagonized the tight binding of Tet3 to paternal chromatin when ERK1/2 was inhibited.In conclusion, we propose that zygotic H3K9me2 and 5mC are regulated by fertilization-dependent ERK1/2, which contributes to the development competence of pre-implantation embryos in mice.The online version contains supplementary material available at 10.1186/s13578-022-00758-x. The paternal and maternal pronuclei show asymmetric epigenetic marks , chromatIn mammals, mitogen-activated protein kinases (MAPKs) regulate the stability of the maternal and zygotic transcripts  and are H3K9me2 is associated with the compact chromatin and the transcriptionally repressive state , 18. It 5mC is one of the well-documented epigenetic factors associated with gene silencing and plays an important role in facilitating the propagation of cellular identity through cell divisions , 25. MeaTherefore, in the present study, we examined the role of zygotic ERK1/2 during preimplantation development and reported the fine regulation of paternal 5mC oxidation and H3K9me2 asymmetry by ERK1/2, which provide insights into the role of ERK1/2 during zygotic reprogramming.We first analyzed the expression of MAPKs during early embryo development by re-analysis the RNA-seq datasets of mouse embryos. As shown in Fig. Oct4 (p < 0.05), Nanog (p < 0.05), Sox2 (p < 0.05), and Klf4 (p < 0.05) were all highly expressed in ERK1/2-inhibited blastocysts  Fig. A, B, sugSince the pronuclear localization of G9a and Tet3 was both increased after ERK1/2 inhibition, and the GDC-0994 concentration required for accumulation of paternal Tet3 was lower than that of paternal G9a, we further assessed the interaction between G9a and Tet3. Under the TP conditions, we found reduced tight binding of Tet3 to chromatin following DMOG treatment, especially in paternal pronuclei (p < 0.01) Fig. A, B. ConTo validate the interaction between Tet3 and G9a, we detected the level of paternal 5mC and 5hmC signals in the presence of both ERK1/2 and Tet3 inhibitors. As shown in Fig. In the present study, we found that fertilization-dependent ERK1/2 is the critical regulator for early embryo development in mice. We further reported reduced asymmetries of 5mC, 5hmC, 5caC, and H3K9me2 at the PN stage after ERK1/2 inhibition. In addition, ERK1/2 inhibition led to excess pronuclear localization of G9a, which subsequently antagonizes the tight binding of Tet3 to paternal chromatin, providing crucial insights into the regulation of DNA methylation and histone modifications crosstalk by ERK1/2.Previous studies revealed that ERK1/2 protein levels increase after fertilization , 43, 44,It was demonstrated that mouse minor ZGA occurs at the PN3 stage, during which, the first zygotic transcription is promiscuous  and the Oct4 and Nanog, which serve as hubs in the core pluripotency network, was increased in the expanded and hatched blastocysts after zygotic ERK1/2 inhibition, suggesting that the regulation of ERK on the determination of the embryonic fate occurs in the first cell cycle, consistent with the results of directing fate specification in the preimplantation embryo by ERK [A recent study revealed that ERK is related to transcription activity and the fluctuation or persistence of ERK signal would irreversibly change the expression of pluripotency genes . In the o by ERK .The level of the 5hmC signal was increased in both paternal and maternal pronuclei at the PN3 stage following the treatment with a low concentration of ERK1/2 inhibitor. A previous study reported the suppression of Tet1 by the ERK pathway in cells . HoweverG9a is essential for preimplantation development , 54 and Our study shows that G9a plays a vital role in 5mC oxidation during paternal genomic reprogramming. The excess pronuclear localization of G9a antagonized the tight binding of Tet3 to paternal chromatin and effectively blocked the accumulation of paternal 5hmC in both ERK1/2 and G9a-inhibited zygotes, suggesting that fertilization-dependent ERK1/2 promotes rapid oxidation of paternal 5mC by inhibiting G9a pronuclear localization. The catalytic activity of G9a has previously been reported that essential for protecting the maternal genomic methylation from Tet3-mediated 5mC oxidation . InteresOur study also reveals the crosstalk between 5mC and H3K9me2 in the zygotic genome. In our ERK1/2 inhibition system, Tet3 converts de novo 5mC into 5hmC, while the deposition of H3K9me2 prevents the accumulation of de novo 5mC in the zygotic genome. Recently, the particular epigenetic landscapes show that 5mC is largely independent of H3K9me2 in differentiated  and diseWe have demonstrated that the inhibition of zygotic ERK1/2 causes excess deposition of H3K9me2 and accumulation of 5mC and its oxides in paternal pronuclei by triggering the pronuclear localization of G9a and Tet3, respectively. In conclusion, we propose that zygotic reprogramming is regulated by fertilization-dependent ERK1/2, which contributes to the development competence of pre-implantation embryos in mice.2.Sperm was obtained from ICR males aged 10–20 weeks. The cauda epididymis was cut open with the tip of syringes to allow sperm swimming out. A mass of sperm was put into Human Tubal Fluid (HTF) fertilization medium supplemented with 4 mg/ml bovine serum albumin  and incubated for 1–1.5 h at 37 °C in 5% COICR females aged 4–6 weeks were superovulated by intraperitoneal injection of 10 U pregnant mare’s serum (PMS) and 10 U of human chorionic gonadotropin (HCG) 48 h later. Cumulus oocyte complexes collected 14 h post HCG injection were incubated with capacitated sperm for 2.5 h. Tet3 inhibition was performed by supplementing with 1 mM dimethyloxallyl glycine  in fertilization medium; oocytes were incubated with DMOG for at least 40 min before addition of sperm.Zygotes were washed off the excess sperm and granulosa cells at 2.5 hpf, and were randomly divided into several equal parts for different treatments: GDC-0994 , U0126-EtOH , SB203580 , SP600125 , BIX-01294 , DMOG , or Decitabine , and collected at times indicated. Zygotes incubated with the matching concentration of dimethylsulphoxide  were served as the controls. Zygotes at early and late PN3, PN5 stages, mitotic phase 1- and 2-cell embryos were collected at 5.5, 8, 11, 17, and 24 hpf, respectively. For preimplantation embryos culture, zygotes were incubated with GDC-0994 for 8.5 h and subsequently transferred into KSOM medium without GDC-0994 for further culture to embryonic day E4.Zygotes were fixed in 3.7% paraformaldehyde (PFA) for 60 min, and permeabilized in PBS, 1% BSA, 0.5% Triton X-100 for 25 min at room temperature (RT). Zygotes were blocked for 1 h in PBS, 1% BSA at RT and incubated with the following primary antibody: H3K9me2 , G9a , and Tet3  overnight at 4 °C. Zygotes were then incubated with Alexa Fluor 488- or 555-conjugated IgG secondary antibody  for 1 h in dark. DNA was stained by 1 μg/ml Hoechst33342  for 15 min, and zygotes were mounted in Vectashield (Vector laboratories) and were visualized using an LSM710 confocal laser scanning microscope (Carl Zeiss) with a × 40 objective.2, 0.5% Triton X-100, 300 mM sucrose in 25 mM HEPES pH 7.4) for 45–60 s until the perivitelline space was eliminated, followed by PFA fixation.TP conditions are used for Tet3 staining in zygotes. Triton pre-extraction was performed as previously described with minor modifications , 31. BriAfter PFA fixation and permeabilization, zygotes were treated with 4 M HCl for 10 min at RT. Zygotes were then incubated in Tris–HCl 100 mM for 10 min, and washed in PBS, 1% BSA, 0.05% Tween20 three times for 15 min. The rinsed zygotes were blocked for 1 h in 1% BSA, 0.05% Tween20 in PBS at RT. Zygotes were then incubated with 5mC , 5hmC  or 5caC  antibody overnight at 4 °C, and subsequently incubated with Alexa Fluor 488- or 555-conjugated IgG secondary antibodies (1:200) for 1 h in dark at RT. DNA was stained by 1 μg/ml Hoechst33342 for 15 min, and zygotes were mounted in Vectashield and imaged as described above.MEK1/2 , phospho-MEK1/2 , ERK1/2 , phospho-ERK1/2 , p38 , phospho-p38 , SAPK/JNK , phospho-SAPK/JNK , Dnmt1 , Dnmt3a , and beta Actin  protein expression in zygotes were verified by western blot analysis. 48–150 mouse zygotes per group were placed in ice-cold RIPA lysis buffer  containing 1% PMSF . The protein was added with 5× SDS-PAGE Sample Loading Buffer  and then denatured on a PCR instrument at 100 °C for 10 min. Proteins were separated by SDS-PAGE (GenScript) at 130 V for 60 min and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes . After transfer, membranes were blocked with TBST that contained 5% BSA for 2 h, followed by incubation at 4 °C overnight with a rabbit primary antibody. After washing three times in TBST (5 min each), membranes were incubated with Horseradish peroxidase-labeled anti-rabbit IgG secondary antibody  for 2 h at RT. Finally, the specific proteins were visualized using Western blotting detection kit (Advansta) and analyzed by ImageJ software. After detection of phosphorylated antigen, the membrane was incubated with membrane regeneration solution (Solarbio) for 60 min for stripping and reprobing of the immunoblot of the total protein levels in the same sample.RNA-seq data of mice (GSE98150) were downloaded from Gene Expression Omnibus. Gene expression of MAPKs during early embryo development was normalized with log2(count + 1).ΔΔct method, and GAPDH was used for normalization as endogenous reference genes.Blastocysts (30 for each treatment) were collected at day E4, and mRNA was purified using Dynabeads mRNA DIRECT™ KIT (invitrogen) following the manufacturer’s instructions. Random primed reverse transcription was performed using HiScript III RT SuperMix with gDNA wiper . cDNA was added to 10 µl of quantitative PCR mix . RT-PCR reactions were performed on a Step-One Plus Real-Time PCR system . The primers for quantitative analysis are shown in Additional file Images were analyzed using ImageJ software. The midsection of each pronucleus was identified using Hoechst33342 staining and determined by the maximal area. The midsection was used to quantify the total intensity following the subtraction of the signal corresponding to the cytoplasmic area (representing staining background). Statistical analysis was carried out using two-tailed Student’s t-test with Welch’s correction when required, using GraphPad Prism software. For gene expression analysis, statistical analysis was performed using two-tailed unpaired t-test. At least three biological replicates were performed for each analysis. Each replicate was conducted by an independent experiment at different times.Additional file 1: Figure S1. Phenotype of  zygotes following treatment with inhibitors of MEK1/2, ERK1/2, p38, and JNK. Figure S2. The expression of phosphorylated and total MAPKs proteins in zygotes treated with corresponding inhibitors. Figure S3. The expression of pluripotent genes in morula that zygotic ERK1/2 inhibited. Figure S4. The expression of Dnmt1 and Dnmt3a in DNMT-inhibited zygotes.Additional file 2: Table S1. Primers used for qRT-PCR analysis.


Bacillus cereus was isolated from the Saudi Red Sea coast and identified based on culture features, biochemical characteristics, and phylogenetic analysis of 16S rRNA sequences. EPSR3 was a major fraction of exopolysaccharides (EPS) containing no sulfate and had uronic acid (28.7%). The monosaccharide composition of these fractions is composed of glucose, galacturonic acid, and arabinose with a molar ratio of 2.0: 0.8: 1.0, respectively. EPSR3 was subjected to antioxidant, antitumor, and anti-inflammatory activities. The results revealed that the whole antioxidant activity was 90.4 ± 1.6% at 1500 µg/mL after 120 min. So, the IC50 value against DPPH radical found about 500 µg/mL after 60 min. While using H2O2, the scavenging activity was 75.1 ± 1.9% at 1500 µg/mL after 60 min. The IC50 value against H2O2 radical found about 1500 µg/mL after 15 min. EPSR3 anticytotoxic effect on the proliferation of (Bladder carcinoma cell line) (T-24), (human breast carcinoma cell line) (MCF-7), and (human prostate carcinoma cell line) (PC-3) cells. The calculated IC50 for cell line T-24 was 121 ± 4.1 µg/mL, while the IC50 for cell line MCF-7 was 55.7 ± 2.3 µg/mL, and PC-3 was 61.4 ± 2.6 µg/mL. Anti-inflammatory activity was determined for EPSR3 using different methods as Lipoxygenase (LOX) inhibitory assay gave IC50 12.9 ± 1.3 µg/mL. While cyclooxygenase (COX-2) inhibitory test showed 29.6 ± 0.89 µg /mL. EPSR3 showed potent inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci. The exposure times of EPSR3 for the complete inhibition of cell viability of methicillin resistant S. aureus was found to be 5% at 60 min. Membrane stabilization inhibitory gave 35.4 ± 0.67 µg/mL. EPSR3 has antitumor activity with a reasonable margin of safety. The antitumor activity of EPSR3 may be attributed to its content from uronic acids with potential for cellular antioxidant and anticancer functional properties.A strain of  Bacterial exopolysaccharides are extracellular organic macromolecules that play a significant role in various bacterial cellular activities, including phage protection, bacterial cell clustering under osmatic stress, and surface adhesion . The exoLactobacillus, Lactococcus, Bifidobacterium, Leuconostoc, Pediococcus, Streptococcus, Enterococcus and Weissella sp.  × 100%, where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells [For cytotoxicity assay, the cells of MCF-7 cells (human breast carcinoma cell line), PC-3 cells (human prostate carcinoma cells), and T-24 (Bladder carcinoma) were seeded in a 96-well plate at a cell concentration of 1 × 10ed cells . EPSR3 and the reference compound (Ibuprofen) were tested to examine the anti-inflammatory response by inhibiting the LOX enzyme from Glycine max (type I-B). This assay was performed according to Granica et al. . The inhInhibitory activity (%) = (1 − As/Ac) × 100, where As is the absorbance in the presence of test substance and Ac is the absorbance of control.50 values—the inhibitory concentration of the samples required to decrease the enzyme’s activity by 50%—were determined from the plotted graphs of enzyme inhibition (%) against the concentrations of the samples.IC50).EPSR3 was evaluated at concentrations ranging from 125 to 0.98 g/mL to assess the anti-inflammatory response induced by inhibiting the COX-2 enzyme. With minor adjustments, this experiment was carried out according to ,61. The The membrane stabilizing activity of EPSR3 was assessed using hypotonic solution-induced erythrocyte hemolysis, according to . The per50 value was defined as the concentration of the sample to inhibit 50% RBCs hemolysis under the assay conditions.% Inhibition of hemolysis (membrane stabilization %) = 100 × {OD1 − OD2/OD1} Where: OD1 = Optical density of hypotonic-buffered saline solution alone OD2 = Optical density of test sample in hypotonic solution. The ICStaphylococcus aureus and coagulase-negative staphylococci were isolated from human beings and belong to the microbiological laboratory collection of the microbiology laboratory of Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Saudi Arabia. Identification and antimicrobial susceptibility of the isolated strains were performed by a VITEK automated system . Nutrient agar was inoculated with this suspension of the respective organism and poured into a sterile petri dish. The agar diffusion assay was performed according to a modified Kirby–Bauer disc diffusion method. One loopful of each test organism was suspended in 3 mL 0.9% NaCl solution separately. All clinical isolates of methicillin resistant and sensitive 8 cfu/mL of bacteria spread on nutrient agar (NA). The discs (6 mm in diameter) were impregnated with 5 mg/disc and placed on the inoculated agar. Negative controls were prepared using the same solvent employed to dissolve extract. Oxicillin (30 μg/disc) was used as positive reference standards to determine the sensitivity of one strain/isolate in each microbial species tested. The inoculated plates were incubated at 37 °C for 24 h for clinical bacterial strains. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms.The EPSR3 were dissolved in dimethylsulfoxide (DMSO) to a final concentration of 30 mg/mL and sterilized by filtration by 0.45 µm Millipore filters. Antistaphylococci tests were then carried out by disc diffusion method  using 10t-test. Only the probability P ≤ 5% was regarded as indicative of statistical significance.The variations between experiments were estimated by standard deviations, and the statistical significance of changes was estimated using the student’s Bacillus strain isolated from the Red Sea Saudi coast produced EPSR3 at (7.95 g/L). From 16S rRNA analysis, the strain was related to B. cereus strain AG3. The EPS displayed moderate scavenging activity on superoxide, hydroxyl radical, and DPPH radicals. In addition, the EPS exhibited a significant protective impact on cancer cell lines. Based on the chemical analysis and in vitro assessments, this study suggests that the EPS from marine B. cereus might contribute to a potential application as a natural antioxidant agent and a new therapeutic agent for treatment of staphylococcal infection and cancers diseases. In this study, a marine


Macular edema recurred in an average of 4.44 ± 0.51 months after treatment, and those patients received combined treatment again. During follow-up, the most common adverse reactions were subconjunctival hemorrhage and increased intraocular pressure, with the incidence of 22% (11/50) and 18% (9/50) respectively. In all cases, the increased intraocular pressure could be controlled by a single intraocular pressure reducing drug. No patient needed to receive anti-glaucoma surgery. The overall incidence of lens opacity was 4% (2/50). After the first injection, no case showed lens opacity. After re-injection, 2 patients (2 eyes) (8%) developed lens opacity. None of the patients showed serious ocular adverse reactions or systemic complications such as vitreous hemorrhage, retinal detachment, endophthalmitis, uveitis or ocular toxicity. The simultaneous vitreous injection of dexamethasone implant and ranibizumab can significantly improve the visual acuity and anatomical prognosis in macular edema secondary to central retinal vein occlusion (CRVO-ME) patients, exhibiting good safety and effectiveness.The purpose of this study was to determine the safety and effectiveness of simultaneous vitreous injection of dexamethasone implant and ranibizumab on macular edema secondary to central retinal vein occlusion (CRVO). We conducted a 6-month retrospective self-control study. Twenty-five patients diagnosed with macular edema secondary to CRVO were enrolled in this study. The patients received intravitreal injection of dexamethasone implant and ranibizumab. The changes in best corrected visual acuity (BCVA), central retinal thickness (CRT) and interocular pressure (IOP) before and at 2w, 1, 2, 3, 4, 5, 6 m after injection were recorded and compared. The adverse reactions in eyes and whole body were observed. The BCVA of all patients at 2 w (61.8 ± 5.42), 1 m (68.68 ± 5.23), 2 m (70.8 ± 5.8), 3 m (68.44 ± 5.61), 4 m (65.76 ± 5.76), 5 m (67.08 ± 5.57), and 6 m (70.12 ± 5.46) after surgery were significantly higher than that before surgery (52.2 ± 5.06, Retinal vein occlusion (RVO) is a retinal vascular disease common in middle-aged and elderly people. The prevalence rate of RVO in people over 40 years old is 1–2% , which iInclusion criteria: : Age> 18This study was approved by the Ethics Committee of the Jinan Second People’s Hospital and followed the “Declaration of Helsinki” (Registration No. JNEYLL201912006).This study was a retrospective case series study, and entailed a review of the medical records of eligible subjects. Fifty-six patients who were diagnosed with ME secondary to CRVO and received simultaneous injection of dexamethasone implant  and ranibizumab  were collected at the Department of Ophthalmology of Jinan Second People’s Hospital from January 2020 to April 2021. Among the 39 patients who received retreatment, there were 25 patients (25 eyes) met the inclusion criteria. Including 11 cases of ischemic type and 14 cases of non-ischemic type. There were 14 male and 11 female patients, with an average age of 54.47 + 7.89 years, ranging from 36 to 70 years old. There were 13 cases with right eye, 12 cases with left eye, and 3 cases with intraocular lens . DexametAll patients received a comprehensive eye examination. The best corrected visual acuity (BCVA) examination was performed using the Early Treatment Diabetic Retinopathy Study (ETDRS) eye chart. The non-contact tonometer  was used to measure the intraocular pressure (IOP) of the affected eye. An OCT instrument  with central retinal thickness (CRT) ≥ 300 μm was used to observe the macular fovea CRT thickness within the range of 1 mm.The intravitreal injection operations were all performed in a sterile laminar flow operating room by the same experienced physician. Lacrimal passage was flushed to rule out dacryocystitis at 3 days before the operation, and levofloxacin eye drops were applied to the operation eye 4 times a day. The pupils were dilated with 0.5% compound tropicamide eye drops at 20 min before injection. Before injection, routine conjunctival sac washing and eye disinfection were performed on the operation eye. Proparacaine hydrochloride eye drops were used for surface anesthesia. After disinfection, the conjunctival sac was rinsed with normal saline. A needle was inserted 3.5 mm behind the superior nasal limbus to inject 0.05 ml (0.5 mg) of Lucentis into the vitreous cavity. The needle was quickly withdrawn and a sterile cotton swab was used to compress the injection site to prevent vitreous incarceration and drug reflux. After 5 min, a 22G special syringe was used to inject 0.7 mg of dexamethasone implant (Ozurdex) into the vitreous cavity at 3.5 mm behind the superior temporal corneal limbus. After the needle was withdrawn, a sterile cotton swab was used to pressurize the injection site to prevent vitreous incarceration. At the same time, the floatation status of the implant was checked. When the intraocular pressure checked by finger palpation was normal, the tobramycin dexamethasone ointment  was applied in the conjunctival sac and the operation eye was covered with sterile gauze.On the first day after the injection, the patient was observed for post-injection reactions, and the BCVA and intraocular pressure were measured. Regular follow-up visits were performed at 2 weeks (2w), 1 month (1 m), 2 months (2 m), 3 months (3 m), 4 months (4 m), 5 months (5 m), and 6 months (6 m) after injection; during the follow-up, BCVA, intraocular pressure, fundus color photography and OCT examination were performed with the same equipment and method as before. If fluorescein fundus angiography (FFA) examination found non-perfusion area in the retina, the laser photocoagulation treatment would be conducted on the peripheral retinal area. During follow-up, if CRT was found to increase by more than 50 um from the lowest recorded level, or visual acuity decreased by 6 letters, the combined injection of vitreous dexamethasone implant and ranibizumab was performed again.p < 0.05 was considered statistically significant.SPSS 23.0 statistical software was used for statistical analysis. The measurement data was expressed as mean ± SD (x±sd) and tested by Shapiro-Wilk to see if the data was normally distributed. Comparison between normally distributed data used One-way repeated measures ANOVA. Non-normally distributed data was tested by paired-sample Wilcoxon rank sum test. At 2 months after operation, the intraocular pressure results of 25 patients were non-normally distributed, the data was expressed in quartile, and the rest of the measurement data were all normally distributed. The specific results of BCVA, CRT, and IOP changes are shown in p < 0.01, p < 0.01).The BCVA change of 25 patients at each time point of 2w, 1, 2, 3, 4, 5, and 6 m after surgery is shown in p < 0.01, p < 0.01).The CRT change of 25 patients at 2w, 1, 2, 3, 4, 5, 6 m after surgery is shown in Among these patients, macular edema (ME) recurred in an average of 4.44 ± 0.51 months after treatment. The patients with ME recurrence received a second combined injection of dexamethasone implant and ranibizumab.In this study, 11 cases showed subconjunctival hemorrhage at the injection site (10 eyes), overall incidence was 22% (11/50), of which 5 cases showed subconjunctival hemorrhage after the first injection , and 6 cases showed subconjunctival hemorrhage after re-injection . All subconjunctival bleeding could be absorbed by itself without treatment.The IOP change of 25 patients at 2 w, 1, 2, 3, 4, 5, 6 m after surgery is shown in A total of 9 cases (7 eyes) had increased postoperative intraocular pressure after treatment, overall incidence was 18% (9/50), of which 6 cases had an intraocular pressure ≥21 mmHg after the first injection, accounting for 24% (6/25), and 4 cases had an increase of ≥10 mmHg compared to before injection, accounting for 16% (4/25). After re-injection, 3 patients had an intraocular pressure ≥21 mmHg, accounting for 12% (3/25); 1 patient had an increase of ≥10 mmHg, accounting for 4% (1/25). In all cases, a single intraocular pressure-reducing drug was enough to control intraocular pressure, and no patients showed secondary glaucoma or needed surgical treatment. The overall incidence of lens opacity was 4% (2/50). After the first injection, no case showed lens opacity. After re-injection, 2 patients (2 eyes) (8%) developed lens opacity, which was subcapsular opacity, and no surgical treatment was required. In addition, no patient showed severe ocular adverse reactions such as vitreous hemorrhage, retinal detachment, endophthalmitis, uveitis, or ocular toxicity, and no systemic complications such as cardiovascular and cerebrovascular diseases were observed .The pathogenesis of RVO-ME is a complex process, in which inflammatory factors and vascular endothelial growth factor play an important role . At presWe compared the improvements of BCVA and macular edema after treatment with the results before surgery, and the difference was obvious. Compared with the preoperative level (52.2 ± 5.06 letters), the average BCVA of the treated eye was already greatly improved at 2 weeks after injection, and was significantly better than pre-operative level at all time points after injection. The BCVA increased most significantly at 2 months after surgery, with an average increase of 18.6 letters compared to the preoperative period ; Table 2The above results suggest that the reason why the patients had better functional and anatomical improvements after surgery might be the synergistic effect from different mechanisms of dexamethasone intravitreal implants (anti-inflammatory drugs) and ranibizumab (anti-VEGF drugs), which resulted in faster, more pronounced and longer BCVA growth and improvement in macular edema. Previous studies have shown that the greatest effect of dexamethasone intravitreal implants (the peak effect) occurs around 2 months after injection , which iThe common adverse events in this study were subconjunctival hemorrhage and glucocorticoid-related intraocular pressure increase, which were consistent with the findings from Li, Haller, et al. . The casThe results of this study showed that the simultaneous injection of dexamethasone implant (Ozurdex) and ranibizumab (Lucentis) into the vitreous cavity could significantly improve the visual acuity and anatomical prognosis of CRVO-ME patients. Moreover, no serious complications occurred in these patients. Therefore, the combined application of anti-VEGF and anti-inflammatory drugs played a complementary and synergistic role, showing good effectiveness and safety.This study is a single-center study, which has some limitations such as small sample size and short follow-up time. Moreover, the patients with branch retinal vein occlusion were not included. In the future, a multi-center, prospective, and randomized controlled study with large sample size is needed to provide more evidence.


EGFR-mutated non-small cell lung cancer (NSCLC).Preclinical evidence suggests the feedforward cytokine loop of interleukin-6/Janus kinases (JAK)/STAT3 plays a role in epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) resistance in EGFR-mutated NSCLC. After erlotinib lead-in , momelotinib was combined and dose escalated in a 3 + 3 study design. The primary endpoint of maximum tolerated dose (MTD) of momelotinib was determined based on the incidence of dose-limiting toxicities (DLTs) during the first 28-day cycle. Secondary endpoints included efficacy and pharmacokinetics (PK).In this phase 1b study, the JAK1/2 and TANK-binding kinase 1 (TBK1) inhibitor momelotinib was evaluated in combination with erlotinib in patients with EGFR TKI-naive, Eleven patients were enrolled across 3 dose levels of momelotinib . The MTD was momelotinib 200 mg QD in combination with erlotinib. Two DLTs of grade 4 neutropenia without fever and grade 3 diarrhea occurred at momelotinib 100 mg BID. Most common treatment-emergent adverse events included diarrhea, dry skin, fatigue, and decreased appetite; the vast majority being grades 1–2. The overall response rate was 54.5%  and median progression-free survival was 9.2 months (90% CI 6.2–12.4). Momelotinib did not affect the PK of erlotinib.EGFR-mutated NSCLC.The JAK1/2 and TBK1 inhibitor momelotinib in combination with erlotinib did not appear to enhance benefit over the historical data of erlotinib monotherapy in patients with NCT02206763.The online version contains supplementary material available at 10.1007/s00280-021-04369-0. EGFR)-mutated non-small cell lung cancer (NSCLC) is rapidly changing. First-  and second-generation  EGFR tyrosine kinase inhibitors (TKIs) were routinely used in the frontline setting until the development of third-generation TKI osimertinib  < 500/μL) and grade 4 thrombocytopenia . Important exceptions for related grade 3 nonhematologic toxicities included grade 3 nausea or vomiting ≤ 48 h duration, diarrhea or rash that improved to grade 0 to 1 within 21 days of interruption of erlotinib, and alanine aminotransferase (ALT) or aspartate aminotransferase (AST) ≥ 5 times the upper limit of normal (ULN) that improved to ≤ 3 times the ULN within 21 days. Treatment delays of ≥ 28 days due to a treatment-emergent AEs (TEAEs) were also considered DLTs. In addition, the treating investigators could deem a TEAE a DLT if in their opinion the TEAE was of potential clinical significance such that further dose escalation would have exposed patients to an unacceptable risk.Imaging by computed tomography (CT) with contrast was performed at baseline, every 8 weeks during year 1 and every 12 weeks thereafter. If a patient had brain metastases, brain magnetic resonance imaging (MRI) was repeated on the same schedule. Secondary endpoints included overall survival (OS), progression-free survival (PFS), and overall response rate (ORR)  as assessed per RECIST version 1.1 .® software .Prior to momelotinib dosing, blood samples were collected before a dose of erlotinib and at 0.5, 1, 2, 4, 6, 8, and 24 h after erlotinib dosing. On day 15 of cycle 1, blood samples were collected at the same time points after dosing with momelotinib and erlotinib (the 24-h time point was only applicable for momelotinib QD dosing). The concentrations of momelotinib and its major metabolite GS-644603 were evaluated using a liquid chromatography-tandem mass spectrometry (LC–MS/MS) assay with solid phase extraction validated over the range of 0.5–1000 ng/mL for each analyte. The concentration of erlotinib was determined using an LC–MS/MS assay with protein precipitation validated over the range of 5–5000 ng/mL. Both assays demonstrated requisite accuracy (± 15%) and precision (coefficient of variation [CV] < 15%). Pharmacokinetic (PK) parameters were estimated using standard noncompartmental methods with Phoenix WinNonlinAny patient who received ≥ 1 dose of study drug was included in the safety and efficacy analyses. Descriptive statistics were used to summarize study characteristics, DLTs, and AEs, including by dose level. ORR was summarized with corresponding two-sided 90% exact confidence intervals (CIs) using the binomial distribution. Both OS and PFS were summarized using Kaplan–Meier estimates and corresponding 90% CIs. Patient concentration data and PK parameters for erlotinib, momelotinib, and its major metabolite GS-644603 were summarized by dose level using descriptive statistics.EGFR-mutated NSCLC were enrolled (Table n = 7) and of Asian and White race . The median time since diagnosis of NSCLC was 1.8 months (range 1.3–2.9), as patients were permitted to be on erlotinib for up to 45 days prior to enrolling on the study. Two patients had undergone prior palliative radiotherapy to bone metastases.A total of 11 patients with treatment-naive, ed Table , includin = 2) and interruptions (n = 5), the average daily dose of momelotinib among patients enrolled at dose level 2A (200 mg QD) and 2B (100 mg BID) was lower than the assigned dose: 178.2 ± 27.1 mg and 186.7 ± 26.5 mg, respectively. The 2 dose reductions of momelotinib were due to AEs (1 each in dose level 2A and 2B). All-cause AEs leading to dose interruption or modification of momelotinib were diarrhea (n = 2 patients), pericardial effusion (n = 1), dyspnea (n = 1), acute myocardial infarction (n = 1), pneumonitis (n = 1), and myalgia, abdominal discomfort, and fatigue (n = 1). All patients were taking erlotinib 150 mg QD at the start of cycle 1, except 1 patient on erlotinib 100 mg QD in dose level 1. There were also dose reductions of erlotinib performed in 4 patients (n = 4), across all dose levels and all for AEs, including 1 to 50 mg.The median duration of treatment exposure to momelotinib was 40.1 weeks (range 2.4–63.1) with a median number of 10 cycles received (range 0.6–15.8). The median duration of exposure to erlotinib was similar at 40.3 weeks (range 2.6–66.3) with a median of 10.1 cycles received (range 0.6–16.6). As a result of dose reductions (Momelotinib was discontinued for progressive disease (PD) in 7 (63.6%) patients, AEs in 3 (27.3%) patients , and per patient discretion in 1 (9.1%) patient. Erlotinib was discontinued due to PD in 7 (63.6%) patients, AEs in 2 (18.2%) patients (1 each of grade 4 neutropenia and grade 1 hepatitis B), and per investigator or patient decision in 1 patient each.n = 3). All patients were taking erlotinib 150 mg QD in dose level 2A at the start of cycle 1, with 1 patient requiring a dose reduction to 100 mg QD on day 14 due to an erlotinib-related toxicity.DLTs were observed in 2 of 5 patients at dose level 2B (momelotinib 100 mg BID). The DLTs were related to both erlotinib and momelotinib. One patient had a DLT of grade 4 neutropenia without fever at day 16 that resolved without treatment on day 19 but resulted in permanent discontinuation of both drugs. The second patient had a DLT of grade 3 diarrhea on day 15 that resolved on day 22, resulting in a momelotinib dose reduction to 150 mg QD with no change in erlotinib dose (150 mg QD). Although there was a DLT exception for grade 3 diarrhea, in light of this toxicity leading to a dose reduction of momelotinib and the other toxicity observed in this cohort, this TEAE was labeled as a DLT due to its clinical significance based on the opinion of the investigators that further dose escalation would have exposed patients to unacceptable risk. There was no DLT observed at dose level 1A (momelotinib 100 mg QD) or dose level 2A (momelotinib 200 mg QD). Based on these findings, the maximum tolerated dose (MTD) was momelotinib 200 mg QD in combination with a standard dosing of erlotinib  had an AE of grade 3 severity or greater, including 3 patients (100%) at dose level 2A and 4 patients (80%) at dose level 2B. There were serious AEs (SAEs) observed in 3 patients (27.3%), including 2 patients in dose level 2A and 1 patient in dose level 2B. Of note, there was no AE of grade 3 severity or greater or SAE observed in dose level 1. There were no treatment-related deaths observed in the study.The most common TEAEs of the combination included diarrhea, dry skin, fatigue, and decreased appetite Table . The mosOf note, a low neutrophil count was observed in 5 (45%) patients, including a grade 3 event occurring in a patient at dose level 2A on day 15 and a grade 4 event occurring in a patient at dose level 2B (previously described DLT); the remaining were grade 1 to 2 in severity and all in dose level 2B. The 3 that were coded as AEs per protocol-specified definition  were considered related to erlotinib and momelotinib, respectively. There were no other recurrent grade 3 or greater hematologic laboratory abnormalities observed. The majority of chemistry laboratory abnormalities were grades 1 to 2, with the most common being AST (72.7%) and ALT increase (54.5%).There were no prespecified AEs of special interest defined in the protocol. However, the following were highlighted: grade 1 peripheral sensory neuropathy in 1 patient at day 142 in dose level 2B; a grade 1 reactivation of hepatitis B in 1 patient that occurred 1 year after start of study treatment, the latter resulting in discontinuation of momelotinib and erlotinib; and no event of cataracts was reported. There was also 1 case of grade 3 pneumonitis considered related to momelotinib at dose level 2B.Confirmed objective responses  were observed in 6 of 11 patients for an ORR of 54.5% (90% CI 27.1–80.0). An additional 4 patients (36.4%) had stable disease and 1 had PD . Responses were observed in all dose levels. Across dose levels, the mean best percentage change in the sum of diameters of target lesions was −42.5 ± 17.1%, with reductions from baseline ranging from 23.1 to 71.7% . The median OS was not reached.max) and a slightly more than dose-proportional increase in exposure (AUCtau)  and combination therapy with momelotinib (day 15 of cycle 1) across all the momelotinib dose level groups. These data suggest there is no impact of momelotinib on the PK of erlotinib. (Supplementary Table 1).Erlotinib exposure as reflected in CEGFR-mutated advanced NSCLC. The MTD was momelotinib 200 mg QD in combination with standard dosing of erlotinib, with QD dosing of 50, 75, 100, and 150 mg permitted. Two DLTs were observed when momelotinib was dosed at 100 mg BID in combination with erlotinib, including grade 3 diarrhea and grade 4 neutropenia. The treatment-related toxicities observed were commonly noted for each agent alone, including diarrhea, nausea, and fatigue for momelotinib [3  were rarely observed with erlotinib monotherapy [KRAS-mutated NSCLC [We conducted a multisite, phase 1b, open-label study of the first-generation EGFR TKI erlotinib in combination with JAK1/2 and TBK1 inhibitor momelotinib in 11 patients with EGFR TKI-treatment–naive elotinib –32 and delotinib , 34. A helotinib , and 6% otherapy , 34. Theed NSCLC .EGFR-mutated NSCLC [max and AUCtau of the major metabolite GS-644603 were several-fold lower than previously reported [KRAS-mutated NSCLC [EGFR-mutated NSCLC, the decision was made to not proceed to the randomized dose expansion of this combination.Although there remains extensive preclinical promise of targeting the JAK/STAT pathway for ed NSCLC , 23–25, ed NSCLC , 34. In ed NSCLC , 34. In ed NSCLC . In addireported –38. Erloreported , which ireported , perhapsed NSCLC . In an eed NSCLC . UnfortuEGFR T790M models [MET amplification [EGFR T790M had an objective response with a median PFS of 4.9 months (95% CI 2.5–7.3), while 2 of 10 patients (20%) without EGFR T790M had an objective response and median PFS of 3.1 months (95% CI 0.0–8.8). There are ongoing studies of third-generation EGFR TKI osimertinib in combination with JAK1 inhibitor itacitinib (NCT03450330 and NCT02917993) in EGFR TKI-resistant NSCLC.Although this study was initiated based on the hypothesis of JAK inhibition preventing resistance to EGFR TKI therapy, there have also been preclinical studies demonstrating the potential utility of combining EGFR TKI therapy and JAK1/2 inhibitors in resistant models . These cM models , 26, 41 fication . This wafication , 22, 41,fication , and frofication , 26, 41.fication , 43. In fication . Five ofEGFR-mutant preclinical models, EGFR inhibition led to feedforward activation of TBK1 and its transcription factor IRF3, which is responsible for type 1 IFN transcription. Targeting the TBK1/IRF3/IFN pathway enhanced sensitivity to erlotinib and afatinib in EGFR-mutant sensitive and T790M xenograft models, respectively. Targeting this pathway also restored sensitivity to erlotinib in non-T790M, non-MET–amplified, EGFR TKI-resistant cell lines. Due to momelotinib’s ability to target TBK1, it was studied in KRAS-mutated NSCLC in combination with the MEK 1/2 inhibitor trametinib [Momelotinib also inhibits TBK1 , 44, whiametinib  and in pametinib . Howeverametinib  and chemametinib .EGFR-mutated NSCLC and did not support enrollment to the randomized dose expansion [EGFR-mutated NSCLC [The combination of the JAK1/2 and TBK1 inhibitor momelotinib with erlotinib did not enhance benefit over historical controls of erlotinib monotherapy in patients with xpansion , 34. Howed NSCLC , 45, 46.ed NSCLC .Supplementary file1 (DOCX 33 KB)Below is the link to the electronic supplementary material.


However, the distribution and translocation of excess Al in citrus species are not well understood.Many citrus orchards of south China suffer from soil acidification, which induces aluminum (Al) toxicity. The Al-immobilization Citrus sinensis (L.) Osbeck] and ‘Shatianyou’ [Citrus grandis (L.) Osbeck], that differ in Al tolerance, were hydroponically treated with a nutrient solution (Control) or supplemented by 1.0 mM Al3+ (Al toxicity) for 21 days after three months of pre-culture. The Al distribution at the tissue level of citrus species followed the order: lateral roots > primary roots > leaves > stems. The concentration of Al extracted from the cell wall (CW) of lateral roots was found to be about 8 to 10 times higher than in the lateral roots under Al toxicity, suggesting that the CW was the primary Al-binding site at the subcellular level. Furthermore, the Al distribution in CW components of the lateral roots showed that pectin had the highest affinity for binding Al. The relative expression level of genes directly relevant to Al transport indicated a dominant role of Cs6g03670.1 and Cg1g021320.1 in the Al distribution of two citrus species. Compared to C. grandis, C. sinensis had a significantly higher Al concentration on the CW of lateral roots, whereas remarkably lower Al levels in the leaves and stems. Furthermore, Al translocation revealed by the absorption kinetics of the CW demonstrated that C. sinensis had a higher Al retention and stronger Al affinity on the root CW than C. grandis. According to the FTIR (Fourier transform infrared spectroscopy) analysis, the Al distribution and translocation might be affected by a modification in the structure and components of the citrus lateral root CW.The seedlings of ‘Xuegan’ [C. sinensis than C. grandis. The aluminum distribution and translocation of two citrus species differing in aluminum tolerance were associated with the transcriptional regulation of genes related to Al transport and the structural modification of root CW.A higher Al-retention, mainly attributable to pectin of the root CW, and a lower Al translocation efficiency from roots to shoots contributed to a higher Al tolerance of The online version contains supplementary material available at 10.1186/s12870-022-03472-5. Acidification of arable soils has increased in China from 1980 to 2000 s , and repCitrus aurantium L.  and ‘Suanyou’ [Citrus grandis (L.) Osbeck] used in the study were identified by Professor Lin-tong Yang of Fujian Agriculture and Forestry University  and deposited as living materials for research purposes in the demonstration orchard of Fujian Academy of Forestry Sciences (FAFS). In December 2018, citrus fruits were harvested under the permission of Professor Xiang-xi Xiao in FAFS and stored in a fridge at 4 ℃. For germination, the seeds of C. sinensis and C. grandis were sown in a plastic tray filled with clean river sand at the greenhouse in early April of 2019. Four weeks after germination, seedlings of uniform size (about 10 cm) were transferred to black tanks containing nutrient solution and aerated for 30 min every two hours. The nutrient solution contained 1 mM KNO3, 1 mM Ca(NO3)2, 0.1 mM KH2PO4, 0.5 mM MgSO4, 10 µM H3BO3, 2 µM MnCl2, 2 µM ZnSO4, 0.5 µM CuSO4, 0.065 µM (NH4)Mo7O24 and 20 µM Fe-EDTA. The pH of the nutrient solution was adjusted to 4.30 using 1 M HCl or NaOH and was replaced every two days. Three months after transplanting, the plants were subjected to the treatments with 0 (Control) or 1.0 mM Al (Al toxicity) in the nutrient solution described above (pH 4.30). The samples of citrus leaves, stems, primary roots and lateral roots were divided and collected 21 days after treatments when visible leaf chlorosis appeared on Al-treated C. grandis leaves.The citrus species ‘Xuegan’ [3/HClO4 , and Al content was quantified according to Hsu [The leaves, stems, primary roots and lateral roots of citrus species were dried and digested in HNOg to Hsu .The crude CW of citrus lateral roots was extracted according to Zhong and Lauchli  with mod4, pH = 4.0) (5 mg CW/1mL solution) in a boiling water bath for one hour and centrifuged at 17,000 g for 10 min for three times to remove the pectin, the resulting pellet (CW-pectin) was pooled and dried. The CW-pectin fraction was extracted by 4% KOH (containing 0.1% NaBH4) or 24% KOH (containing 0.1% NaBH4) under room temperature three times for 24 h in total to further remove the hemicellulose 1 (HC-1) and hemicellulose 2 (HC-2). The pooled pellets were CW-pectin fractions without HC1 (CW-pectin-HC1) and HC2 (CW-pectin-HC1-HC2), respectively. The Al content of the CW and CW fractions  was quantified according to the method described above. The Al content of CW and CW fractions was expressed as mg · g− 1 DW (dry weight) of lateral roots.The dry crude CW from the lateral roots of citrus seedlings was further fractioned according to Yang et al.  Briefly,Cs6g03670.1, Cg1g021320.1, Cs3g18690.1 and Cs6g05460.1) involved in Al translocation according to Wu et al. [2O. The program consisted of initial denaturation at 95 ◦C for 2 min, followed by 40 cycles of 95 ◦C for 10 s, 57 ◦C for 35 s and 72 ◦C for 30 s. Citrus genes actin (Cs1g05000.1) was used as internal references [https://www.citrusgenomedb.org/) and listed in Table −ΔΔCT according to Livak and Schmittgen [Lateral roots of two citrus species were harvested for the relative expression analysis of genes  was pumped by a peristaltic pump at 0.2 mL/min through a 2 mL column loaded with 10 mg root CW for Al adsorption. The solution after CW adsorption was then collected by a fraction collector at 20 min intervals until the Al content was equal to the adsorption solution. The residue Al3+ left in the system was washed by 0.5 mM CaCl2 (pH 4.5) at 0.6 mL/h for 1 h before Al desorption by 2.5 mM CaCl2 (pH 4.30) at 0.2 mL/h until the Al concentration in the collector was below the detection limit. Finally, the Al content in the fraction collector was quantified, and the kinetics were analyzed within 600 min. The Al absorption and desorption kinetics were performed three times independently.The Al adsorption and desorption kinetics were performed according to Zheng et al.  with mod-1 were recorded using Vertex 70 spectrometer with a resolution of 4 cm-1 and 32 scans per sample. The obtained spectra were normalized and baseline-corrected by OPUS management software before being exported to Excel. The data of FTIR spectra were processed by Origin Pro 2020b . The OPLS-DA was performed in SIMCA 14.1 .2 mg dry CW of citrus lateral root was mixed with 200 mg KBr and pressed into a disk by FW-5 A Pressor. The IR spectra of CWs ranging from 4000 − 400 cmP < 0.05) among treatments were statistically evaluated by two-way ANOVA using Duncan’s test, using the SPSS 16.0 . All the values are presented as means ± SE. Figures except OPLS-DA were generated by using Sigmaplot 12.0.Data analysis was performed by two-way analysis of variance, and significant differences , roots (B), stems (C), leaves (D), shoots (E) and ratio of root/shoot (F) of C. sinensis and C. grandis seedlings. Seedlings of C. sinensis and C. grandis were treated with nutrient solution  or supplemented by 0.5, 1.0 and 2.0 mM Al3+ (pH 4.3) for 15 weeks. The values represent mean ± SE (N = 6). Significant differences (p ≤ 0.05) between treatments are indicated by different letters.


IL22 is an important cytokine involved in the intestinal defense mechanisms against microbiome. By using ileum-derived organoids, we show that the expression of anti-microbial peptides (AMPs) and anti-viral peptides (AVPs) can be induced by IL22. In addition, we identified a bacterial and a viral route, both leading to IL22 production by T cells, but via different pathways. Bacterial products, such as LPS, induce enterocyte-secreted SAA1, which triggers the secretion of IL6 in fibroblasts, and subsequently IL22 in T cells. This IL22 induction can then be enhanced by macrophage-derived TNFα in two ways: by enhancing the responsiveness of T cells to IL6 and by increasing the expression of IL6 by fibroblasts. Viral infections of intestinal cells induce IFNβ1 and subsequently IL7. IFNβ1 can induce the expression of IL6 in fibroblasts and the combined activity of IL6 and IL7 can then induce IL22 expression in T cells. We also show that IL22 reduces the expression of viral entry receptors , increases the expression of anti-viral proteins  and, consequently, reduces the viral infection of neighboring cells. Overall, our data indicates that IL22 contributes to the innate responses against both bacteria and viruses. ten Klooster et al. use ileum-derived organoids and report that bacterial and viral stimuli can drive IL22 production by T cells via distinct signaling pathways, and that IL22 can stimulate expression of anti-microbial and antiviral factors. This study sheds light on how enterocytes, fibroblasts and myeloid cells work synergistically to induce IL22 response against both bacteria and viruses. Antibacterial peptides, such as Reg3β and Reg3γ, produced by Paneth cells in the small intestine, ensure that the inner intestinal mucus layer contains little to no bacteria2. The importance of the C-type lectin Reg3γ in this process has been demonstrated by Hooper et al., who show that Reg3γ knockout mice suffer from a high influx of bacteria in the intestinal mucus layer2. In addition, lack of Reg3γ expression in human subjects is highly associated with Inflammatory Bowel Disease (IBD)4.The intestine contains many bacteria which, when given the opportunity, cause local damage and invade the mucus layer and the underlying tissues8. There is data suggesting that the cytokines IL23, IL6, TNFα, and IL1α/β and Serum Amyloid A3 (SAA3) are involved in the induction of IL22 in T cells of different origin8. These cytokines can be induced in myeloid cells, such as macrophages and dendritic cells (DC) by LPS9, a potent activator of the TLR4 pathway10. The combination of IL6, IL1β, and TNFα can induce the expression of IL22 in adult peripheral blood cells when combined with anti-CD3/anti-CD286. SAA3 can induce IL22 expression in bone marrow-derived neutrophils7 and in in vitro-differentiated Th17 cells11.The expression of Reg3γ can be enhanced by IL22, a cytokine that can be secreted by neutrophils, Th22, Th17, ILC3, or γδ T cells, but the regulation of IL22 expression and secretion in these different cell types is not entirely clear12. Besides bacteriophages, the gut also hosts viruses that target eukaryotes, such as Hepeviridae, Polydnaviridae, Tymoviridae, and Virgaviridae families, which have also been associated with the pathogenesis of IBD15.In addition to bacteria, the gut is also home to a diverse population of viruses. Most of these viruses are bacteriophages, which target bacteria and thus affect the bacterial composition in the intestineOur data demonstrates a previously unrecognized pathway of bacterial and viral induced IL22 production by intestinal T cells. By using co-cultures of 2D and 3D intestinal organoids, fibroblasts, macrophages, DC and T lymphocytes, we show that IL6 and TNFα synergize in stimulating the production of IL22 in T cells when stimulated with bacterial products, such as LPS, Pam2, and Pam3. In addition, we show that viral infections of intestinal organoids induce IFNβ1 that, via subsequent autocrine stimulation, induces IL7 expression in enterocytes. Moreover, the secreted IFNβ1 can induce the expression of IL6 in fibroblasts and the combined secretion of IL6 and IL7 can also induce the expression of IL22 in T cells, and thus reduce the expression of viral entry receptors  and enhance the expression of anti-viral genes .2. However, these experiments were performed in vivo and could not exclude the role of immune cells, which are very likely to be involved in this process11. To test whether LPS directly induces expression of Reg3γ in Paneth cells or requires the mediation of immune cells, we stimulated the dendritic JAWSII cells and macrophage RAW264.7 cells with LPS and used the supernatants of these cells to either stimulate EL4 T cells or ileum-derived organoids. Organoids were then analyzed for Reg3β and Reg3γ mRNA expression  can activate Paneth cells, we performed a microarray on ileum-derived organoids and noticed that these organoids express only a small subset of cytokine receptors  from a C57BL/6 mouse and exposed these cells to supernatant from LPS-activated bone marrow-derived DCs. IELs were cultured in the absence of other activating factors, such as anti-CD3 or IL7. We determined PCR Fig.  and ELISPCR Fig. . Indeed,PCR Fig. . SubsequPCR Fig. . The expity Fig. . Similarnes Fig. , whereas4 h Fig. , showing11. To test the involvement of these cytokines in our in vitro system, we analyzed our previously published microarray dataset on RAW264.7 cells which were stimulated with LPS17 and observed the expression of multiple cytokines, including Il23, Il6, Tnfα, and Il1α/β  with or without a capturing antibody for TNFα and determined the IL6 secretion by ELISA Fig. . We obsel1α Fig. . In addil1α Fig. . The TNFl1α Fig. . Overall9. However, intestinal enterocytes are constantly exposed to these bacterial compounds, whereas the intestine is not constantly highly inflamed, suggesting that intestinal epithelial cells (IECs) do not produce high levels of pro-inflammatory cytokines or might express anti-inflammatory cytokines, which could inhibit the inflammatory activity of intestinal myeloid cells. To test which genes are activated in intestinal enterocytes in response to LPS, we seeded ileum-derived organoids either in Matrigel (3D) or on collagen I-coated 48 wells plates (2D)21 and allowed them to grow for 2 days. The different culture systems, 2D vs. 3D, allow us to discriminate between crypt structures (3D), enriched in stem cells and Paneth cells, and more differentiated villi-like cultures (2D)9, containing high amounts of enterocytes and endocrine cells. Subsequently, we stimulated these cultures with LPS, Pam2, or Pam3 for 24 h. From these cultures we isolated RNA to perform microarray analyses. Data analysis on genes involved in inflammatory pathways, revealed that Tnfsf13/April, Saa3, and Tnfα were upregulated after LPS stimulation of organoids cultured in 2D , Tmprss2 (SARS-Cov-1 and 2), Dpp4 (MERS), Tnfrsf14 (HSV), and Cd46 (MV), while increasing the expression of antiviral proteins such as Rsad2, Apobec1 and 3, Irf9, Mx1, Isg20, and AoS  prior to viral infections with MHV-GFP. After 24 and 48 h, cells were fixed and stained for F-actin and nuclei lei Fig.  and the lei Fig. . We obselei Fig. , suggest19, resulting in IL22 secretion in T cells  is induced in the presence of gut microbiota35, and here we found that IFNγ is also able to induce the expression of these AMPs. Remarkably, we also found that IFNγ can inhibit the IL22-mediated upregulation of Reg3β and Reg3γ. These findings emphasize the importance of the specific cellular source of IL22, which in some conditions will be neutrophils, being strict IL22 producers7, and in other conditions intraepithelial T cells, producing IL22 and IFNγ simultaneously  autophagy43 and dengue virus replication44. Consequently, a consensus is beginning to emerge that IL22 may exert antiviral control during infection. Indeed, our microarray analyses show increased expression of antiviral genes, such as Rsad2, Ifit1, 2, and 3 after IL22 exposure  could be reduced with an IL22 pretreatment 55, which can either increase or decrease the entry of a specific virus55, thereby changing the susceptibility to viral infections. Overall, viruses can induce the expression of IL22 through an IFNβ1–IL7–IL6-axis and successively, IL22 can help defend against viral infection on different levels by regulating 1) viral degradation, 2) viral entry, and 3) viral sensitivity through sugar modifications of extracellular proteins.Treatment of organoids with IL22 increases the expression of the fucosyltransferase 2 gene ut2 Fig.  and in vCollectively, here we provide fundamental knowledge that could help in designing selective strategies to improve innate intestinal defense processes preventing bacterial and viral infections and subsequent chronic inflammatory diseases, such as IBD.56 cells were split 1:30 every 3–4 days. NIH3T3 (ATCC® CRL-1658™) were split 1:10 every 3–4 days by scraping with a cell-scraper (Merck CLS3010-100EA).For all cultured cells, we used DMEM F12 + Glutamax medium  supplemented with 10% Fetal Calf Serum  or 5% for EL4 and RAW264.7 cells, 1% Pen/Strep  and 1 mM sodium pyruvate . JAWSII (ATCC® CRL-11904™) cells were grown in the presence of GMCSF  5 ng/ml and split 1:5 every 7 days. EL4 (ATCC® TIB-39™), RAW264.7 (ATCC® TIB-71™), JAWSII (ATCC® CRL-11904) and LR729. Briefly, LR7 cells were cultured in T75 bottles until a confluency of 80%. Subsequently, 100 μl of MHV-GFP recombinant virus suspension was added and incubated for 24 h. The next day, medium was collected and centrifuged for 5 min at 1200 rpm to remove LR7 cells. The virus containing medium was aliquoted and stored at −80 °C.Construction of MHV-2aGFPSRec, which contains an eGFP expression cassette between the 2a and the S genes at the position of the HE pseudogene, was described previouslyg for 5 min between every wash step). A Polter–Elvehjem tube was then used to fragment the tissue and the cell suspension was transferred to a tube with ice-cold PBS, washed three times with PBS and subsequently filtered through a 100 μm filter. The now isolated crypts were spun down at 335 × g for 5 min at room temperature and supernatant was removed. The crypts were resuspended in Matrigel , and drops of 25 μl suspension were added to each well (Corning Costar). The plates were then incubated for 15 min at 37 °C until the droplets had solidified. One milliliter of organoid culture medium supplemented with 10% FCS, 10 μM Y27632 , CHIR 4,3 μM,  and 2-Propylpentanoic acid . After 3 days of incubation at 37 °C and 5% CO2, medium was replaced by normal culture medium: DMEM/F12 medium containing GlutaMAX supplement, 1 mM sodium pyruvate, MEM Non-Essential Amino Acids, 100U/ml Penicillin-Streptomycin, R-spondin 1 (homebrew), WNT (homebrew), and the BMP4 inhibitor DMH1 0.5 µg/ml  to stimulate organoid formation. These organoids were passaged once a week in a 1:4 ratio.A female mouse C57/BL6, 6 months of age was sacrificed using carbondioxide. The intestine was removed and the ileum isolated. Villi were removed from the ileum by scraping with a scalpel and the tissue was cut into small pieces, transferred to a tube with ice-cold PBS and washed three times with PBS . After solidification of the Collagen I coating, a suspension of organoids in medium supplemented with 10% FCS, 10 μM Y27632 , CHIR 4,3 μM,  and 2-Propylpentanoic acid  was added to each well. The organoids were grown for 2 days at 37 °C and 5% CO57. Medium was refreshed after 3 days. DCs were collected and stimulated with 10 ng/ml LPS (Sigma) for 24 h in a 12-wells plate . Supernatant was collected and stored at −80 °C until use.Bone marrow-derived dendritic cells (DC) were isolated from C57BL/6 mice  and cultured in presence of GM-CSF for 6 days according to the methods described in Lutz et al.5 cells/well in a 96-wells round bottom plate (Greiner). Culture supernatant was harvested after 24 h and used to stimulate intestinal organoids. The use of mouse intestine was approved by the local committee for care and use of laboratory animals at Utrecht University. Only surplus mice were used for this study.Small intestine of female C57BL/6 mice (7–10 weeks old) was removed and flushed with phosphate buffered saline  to remove luminal contents. After removing fat tissue and Peyer’s patches, the intestines were cut open longitudinally and the epithelial layer was scraped off. Tissue was collected in RPMI (supplemented with 10% FCS and 1% penicillin/streptomycin) containing 1 mM dithiothreitol (DTT) and incubated for 40 min with gentle rotation. Tissue fragments were washed once, vortexed to obtain single cells and run through a nylon wool column to remove excess epithelial cells and non-T immune cells. The eluates were washed once and a density gradient centrifugation was performed using 40/80% percoll . The cell layer at the 40/80% interface was harvested. These freshly isolated IEL were stimulated with BMDC LPS supernatant in a density of 1 × 10E. coli-derived B4 LPS  0.2 µg/ml for 18 h and supernatant was collected the next day. Subsequently, EL4 cells were seeded in the presence of LPS, JAWSII supernatant or cytokines (Peprotech) as indicated and incubated for 18 h before cytokine and QPCR analysis.JAWSII and RAW264.7 cells  were seeded in the presence of NIH3T3 fibroblast were seeded in 12-wells plates, 300,000 cells/well/3 ml and allowed to adhere for 24 h. Next, when indicated, we added 50,000 RAW264.7 cells and/or 50,000 EL4 cells. Subsequent stimulations were performed for indicated time points and concentrations.Recombinant mSAA3 was produced in HEK293T cells and purified by Ni-beads. Stimulations with hSAA1 (Peprotech 300-53) were performed in the presence of Polymyxin B (Merck 1547007-200MG), which sequesters potential LPS contamination. Recombinant mIL22BP (RnDsystems 2376-BP-025).Blocking antibodies that were used are: anti-mIL6 (Peprotech 500-P56), anti-mIL22 (Peprotech 500-P223), anti-mTNFβ (Peprotech 500-P64), anti-mIL9 (Peprotech 500-P59), anti-mIL7 (Peprotech 500-P57), anti-mIFNγ (Peprotech 500-P119), anti-mIFNγR1 (RnDsystems MAB10262), anti-IFNγR2 (RnDsystems MAB773), and anti-mIL1α (Peprotech 500-P51).2O and 0.5 μM forward and 0.5 μM reverse primer. Reactions were incubated in an Applied Biosystems 7500 Fast Real-Time PCR system in 96-well plates. The primers used are described in Table Hprt, subsequently plotted relative to control.Briefly, RNA from each sample was isolated  and 200 ng of RNA was used for cDNA synthesis by reverse transcription . Real-time PCR reactions included 5 μl of diluted RT product (1:6 dilution), 10 μl FAST SYBR Green buffer , 4.8 μl H59. Probe sets were assigned to unique gene identifiers, in this case Entrez IDs. The probes on the Mouse Gene 1.1 ST arrays represent 21,213 Entrez IDs. Array data were analyzed using an in-house, on-line system60.One hundred nanogram of RNA was used for Whole Transcript cDNA synthesis . Hybridization, washing and scanning of Affymetrix GeneChip Mouse Gene 1.1 ST arrays was carried out according to standard Affymetrix protocols. All arrays of the small intestine were hybridized in one experiment. Arrays were normalized using the Robust Multi-array Average method2SO4. Colorimetric determination was performed in an ELISA plate reader at 450 nm wavelength.Supernatants of exposed cells were collected and diluted 1:1 in ELISA dilution buffer according to manufacturer’s protocol . Briefly, plates were coated with capture antibody for 18 h, blocked for 1 h, washed 3× and exposed for 2 h to 100 μl diluted supernatant from exposed cells. Then plates were washed 3× and detection antibody was added for another 2 h after which the plates were washed again for 3× and streptavidin-HRP was added for 30 min. Plates were washed again and HRP substrate  was added for 10–30 min after which the reaction was stopped by adding 50 μl of 1 M HCultured organoids were fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton-X100 for 5 min and subsequently washed twice with PBS. These organoids were incubated with either anti-IL7 antibody  for 2 h at 37 °C. Alexa Fluor 488 goat anti-rabbit (Life Technologies) and Alexa Fluor 488 donkey anti-mouse (Life Technologies) were used as secondary antibodies. Actin and nuclei were stained using Rhodamine Phalloidin (Life Technologies) and Hoechst 33342 , respectively. Images were acquired on a Olympus IX71 fluorescence microscope using CellSens software (Olympus Corporation).P < 0.05 was considered to indicate statistical significance. For comparison of two groups, we performed an unpaired t-test (two-tailed) with Welch’s correction on Log2 transformed data. All experiments were performed in triplicate and all experiments were executed at least three times, except for the array data, which were performed only once in triplicate.All data is shown as mean ± SD. For multiple conditions, measurements were analyzed on Log2 transformed data by a Welch and Brown–Forsythe test followed by Dunnett’s T3 multiple comparisons test, Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Reporting Summary


Due to the increase of type 2 diabetes (T2D), the number of patients in treatment with multiple anti-diabetic agents is increased. According to the recent recommendation of treatment guidelines, sodium-glucose cotransporter 2 (SGLT2) inhibitors would be used as additional treatment to the currently administered anti-diabetic drugs for poorly controlled T2D patients. Here, we assessed the efficacy of SGLT2 inhibitors added to the current treatment with metformin, dipeptidyl peptidase-4 (DPP4) inhibitors, or both in Japanese T2D patients.Japanese T2D subjects with poor glucose control, who were treated with metformin (n = 10), DPP4 inhibitors (n = 11), or both (n = 28) and who were in need of additional treatment, were recruited. HbA1c levels before and 6 months after addition of SGLT2 inhibitor treatment were used to compare the effectiveness. The HbA1c levels after addition of SGLT2 inhibitors significantly decreased in all groups. The change in HbA1c levels (delta HbA1c) showed no significant difference between the three groups. The present data indicated that addition of SGLT2 inhibitors to metformin and/or DPP4 inhibitors is equally effective in the treatment of Japanese T2D patients. When diet and exercise treatments are not sufficient to obtain good glycemic control in type 2 diabetes (T2D) patients, treatment guidelines recommend initiation of treatment with single oral hypoglycemic agent , 2. A JaHowever, single anti-diabetic oral drug gradually becomes less effective in obtaining good glycemic control, and additional treatment is required. Currently, many Japanese T2D patients undergo combination treatment with DPP4 inhibitors and metformin, which are most commonly used anti-diabetic drugs. Although this combination treatment is effective, its efficacy decreases over time, and a third drug is required to obtain good glycemic control.Sodium-glucose cotransporter-2 (SGLT2) inhibitors are a new class of anti-diabetic drugs and have glucose lowering effects by inhibiting the renal reabsorption of glucose and increasing glucosuria , 6.Considering the pharmacological action of SGLT2 inhibitors, which differs from that of DPP4 inhibitors and metformin, the additional improvement of glycemic control is expected when SGLT2 inhibitors are added to DPP4 inhibitors or metformin. However, it remains unclear whether the effectiveness of SGLT2 inhibitors differs when added to monotherapy or combination therapy of DPP4 inhibitors and metformin in T2D patients.Here, we assessed the efficacy of SGLT2 inhibitors added to the current treatment with DPP4 inhibitors, metformin, or both in Japanese T2D patients.This is a retrospective cohort study including outpatients of Tsukuba Medical Center and Matsumura General Hospital for diabetes.Thirty-four subjects (18 men and 16 women) treated with combination therapy of DPP4 inhibitors and metformin, 11 subjects (6 men and 5 women) treated with DPP4 inhibitors and 10 subjects (4 men and 6 women) treated with metformin were recruited. The length of diabetes in DPP4 inhibitor and metformin combination treated group was 23 subjects for more than 10 years, 5 subjects for 5 years, 3 subjects for 2 years and 3 subjects for a year. The length of diabetes in only DPP4 inhibitor treated group was 3 subjects for more than 10 years, 1 subject for 8 years, 3 subjects for 5 years, 1 subject for 4 years and 3 subjects for 2 years. The length of diabetes in only metformin treated group was 6 subjects for more than 10 years, 1 subject for 9 years, 1 subject for 9 years, 1 subject for 3 years and 2 subjects for 2 years. Some of the subjects recruited in this study had other metabolic disorders (hyperlipidemia and/or hyperuricemia). Three subjects had hyperlipidemia, 1 subject had hyperuricemia and 2 subjects had both for DPP4 inhibitor treatment group. Nine subjects had hyperlipidemia, 0 subject had hyperuricemia and 1 subject had both for the metformin treatment group. Seventeen subjects had hyperlipidemia, 0 subject had hyperuricemia and 4 subjects had both for combination treatment group.All the patients recruited in this study who are using metformin were treated with 500 mg/day. The DPP4 inhibitors used by the subjects in this study were alogliptin, sitagliptin, linagliptin, vildagliptin, anagliptin, saxagliptin, tenegliptin. All DPP4 inhibitors were used in standard dosage according to the drug protocol. Drugs are all administered orally.HbA1c levels were used to assess the effectiveness of adding SGLT2 inhibitors to the current treatment. HbA1c levels were measured before and 6 months after adding SGLT2 inhibitors. SGLT2 inhibitors were added in the patients who had HbA1c levels higher than 6.9 in spite of the DPP4 inhibitors and metformin treatment. Added SGLT2 inhibitors were ipragliflozin, canagliflozin, dapagliflozin, luseogliflozin or empagliflozin. All SGLT2 inhibitors were used in standard dosage according to the drug protocol.t-test was used for two-group comparisons. All statistical tests were two-tailed, with values of 0.05 being considered statistically significant.All data are presented as mean ± SEM. Student’s The average age of the 11 patients treated with DPP4 inhibitors, the 10 patients treated with metformin, and the 34 patients treated with the combination of those two drugs was 56.3 ± 7.4, 53.0 ± 3.2, and 55.1 ± 1.7 years old, respectively, showing no significant difference.The average body mass index of each group were not significantly different: 26.0 ± 2.3 in the DPP4 inhibitor treatment group, 26.8 ± 1.2 in the metformin treatment group, and 26.1 ± 1.2 in the combination treatment group.HbA1c levels before the addition of SGLT2 inhibitors were approximately the same among the three groups; 8.7 ± 0.7% in the DPP4 inhibitor treatment group, 8.6 ± 0.5% in the metformin treatment group, and 8.5 ± 0.3% in the combination treatment group.Addition of SGLT2 inhibitors significantly improved HbA1c levels to 7.6 ± 0.5%, 7.5 ± 0.3% and 7.6 ± 0.2%, respectively , a hormone which stimulate insulin secretion under hyperglycemic condition and contribute to nearly 70% of the post-prandial insulin secretion, being considerably low in both healthy and T2D subjects compared to Caucasian –15. TherOn the other hand, metformin, which improves insulin resistance, is also reported to have a positive effect on Japanese T2D patients and widely used for its high efficacy, low cost and low risk of side effects .A considerable number of Japanese T2D patients are therefore under treatment with DPP4 inhibitors, metformin, or both. The HbA1c lowering effect of DPP4 inhibitors and that of metformin are reported to be similar in Japanese T2D patients , 19.The recent addition of SGLT2 inhibitors widened the selection of anti-diabetic drugs to treat T2D patients. Clinical trials such as EMPA-REG OUTCOME even raised the possibility of positive effects of SGLT2 inhibitors on cardiovascular outcome .Based on the study analyzing the data from Japan Medical Data Centre, it is reported that the probability on remaining on treatment with combination of two different antidiabetic drug for 12 month remain to 40–54% , 21. TheIn this study we assessed the efficacy of SGLT2 inhibitors added to currently administered DPP4 inhibitors, metformin, or both in T2D patients. Our present data showed that additional SGLT2 inhibitors had a similar effect on lowering HbA1c levels in T2D patients treated with DPP4 inhibitor, metformin, or both.The addition of SGLT2 inhibitor on patient treated with combination of DPP4 inhibitor and metformin showed similar effect compared to adding SGLT2 inhibitor on monotherapy group. One possibility is that DPP4 inhibitor and metformin combination group consisted with patients with longer duration of diabetes period (23 subjects were diabetes for more than 10 years). Further study on influence of the diabetes duration is required in the future.The present study indicates that the addition of SGLT2 inhibitors is effective in Japanese T2D patients who are unable to obtain good glycemic control with DPP4 inhibitors or metformin, or both. Therefore, the addition of SGLT2 inhibitors should not be hesitated for glycemic control improvement in such patients.Due to a small sample size of subjects, further study is required. Also, due to the small sample size we were unable to compare the effect of adding SGLT2 inhibitors on different DPP4 inhibitors. Further study is also required.