The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca In pancreatic beta cells, Ca2+ triggers insulin secretion to maintain postprandial blood glucose (2+ is sequestered within the endoplasmic reticulum (ER), the organelle where the synthesis and folding of secretory proteins occurs, along with lipid synthesis  is associated with ER stress and apoptosis and accompanies many pathologies  and Ca2+ release by inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)  . Both IPnditions , 14, 15.nditions ; IP3R1 inditions . RyRs arand RyR3 , with Ryand RyR3 . Beta-ceablished .2+ handling and the production of cytosolic Ca2+ ([Ca2+]cyto) oscillations. We used tunicamycin (TM) to experimentally induce ER stress in the rat insulin-secreting INS-1 832/13 beta-cell line, as well as isolated mouse islets. TM activates the UPR by inhibiting N-acetylglucosamine phosphotransferase, leading to protein misfolding in the ER  and led to the appearance of [Ca2+]cyto oscillations in parallel with oscillations in membrane potential    . In respper)   and dantC, 1 μM) , to blocC, 1 μM) , 23, 24,C, 1 μM) .A–C, TM treatment decreased IP3R1 and IP3R3 mRNAs, while the IP3R2 transcript remained unchanged after a 16 h exposure to TM in INS-1 832/13 cells. We also found that RyR1 mRNA was increased by TM treatment, while the level of RyR2 transcript was unchanged (D and E). While we wished to measure RyRs and IP3Rs at the protein level, our efforts to do so were hampered by the lack of commercially available, working antibodies for these ER Ca2+ channel proteins.We first analyzed the effect of TM on the expression of each of the receptor isoforms. As shown in nchanged , D and E2+ (2+ probe D4ER was transiently expressed in islets and INS-1 832/13 cells with adenovirus. Islets and INS-1 832/13 cells were then treated with vehicle control (DMSO), TM, XeC, Dan, TM+XeC, or TM+Dan for 16 h, and [Ca2+]ER was measured using a recording solution containing 5 mM glucose. Representative traces show [Ca2+]ER  in islets and the effect of the reversible SERCA blocker cyclopiazonic acid  reduced ER Ca2+, as expected . Steady-state [Ca2+]ER is measured in 5 mM glucose in islets (B and C) and in INS-1 832/13 cells (D and E) in the absence of CPA. Exposing islets to TM for 16 h significantly reduced steady-state [Ca2+]ER compared to controls, and the presence of either XeC or Dan with TM prevented ER Ca2+ loss. Interestingly, including Dan with TM slightly increased [Ca2+]ER over control levels but had no effect in the controls, perhaps because ER Ca2+ leak through RyRs was low in the controls . Figure 2F shows representative ER Ca2+ responses to CPA in 5 mM glucose in control or TM-treated islets, and we saw that TM-treated islets had lower ER Ca2+ both before and after CPA acute application compared to control. The change in D4ER arbitrary units was calculated by subtracting the unit in steady state at the end of the recordings after CPA application from the unit at the beginning of the recordings before CPA application. We found that the drop in ER Ca2+ was significantly smaller after TM exposure than control .To test whether blocking IP3Rs or RyRs prevented the TM-mediated reduction of ER Ca2+ , the ER expected   (2+]cyto (2+ oscillations in 5 mM glucose (2+ oscillations in stressed islets (A and C); however, TM-triggered oscillations were suppressed when Dan was present (<5% oscillating islets) (B and D), suggesting RyRs but not IP3Rs were involved in their genesis. In Dan or XeC-treated islets, as for DMSO, little or no oscillatory Ca2+ activity was observed in the absence of TM , TM, XeC, Dan, TM+XeC, or TM+Dan for 16 h before recording cyto oscillations in 5 mM glucose, we transfected RyR1 siRNA in INS-1 832/13 cells, resulting in a 50% decrease of RyR1 mRNA (A). Decreasing RyR1 expression may increase RyR2 mRNA levels, but not significantly (B). IP3R1 mRNA was found to be elevated (C). Transfected cells were recorded using imaging buffer containing 5 mM glucose (D). After 16 h of TM treatment, the percentage of cells displaying [Ca2+]cyto oscillations was decreased in RyR1-knockdown cells (∼10%) compared to siCon cells (∼40%) (E). RyR1 knockdown did not affect the Ca2+ activity of unstressed control cells (D and E).To confirm that RyR1 played a role in the production of TM-triggered cyto oscillations in 5 mM glucose, we silenced RyR2 using siRNA in INS-1 832/13 cells and achieved a ∼50% reduction in RyR2 mRNA (A). Neither RyR1 or IP3R1 mRNA was changed after RyR2 knockdown (B and C). The cytosolic Ca2+ levels of the transfected cells were recorded using imaging buffer containing 5 mM glucose (D). The percentage of cells displaying [Ca2+]cyto oscillations in response to 16 h of TM treatment in this case was unaffected by silencing RyR2 (E).RyR2, rather than RyR1, is the predominant RyR isoform of beta cell in terms of abundance . To studyR2 mRNA A. Neithenockdown , B and C glucose D. The peing RyR2 E.Figure XBP1 reached its peak at this time , ATF4 (B and E), or CHOP (C and F), which we observed when these inhibitors were included with TM. Thus, blocking IP3Rs or RyR1 failed to prevent UPR activation in TM-treated beta cells.Activation of the UPR occurs in response to TM in many cell types, including beta cells , 20, 30.tal XBP1 , A and DD), ATF4 , B and E or CHOP , C and FATF4 and CHOP expression in RyR1, RyR2, or RyR1+2 knockdown cells and observed that neither ATF4 nor CHOP upregulation was reversed by silencing these isoforms individually or collectively (A and B). To further confirm this, we also examined the effect of blocking RyRs on UPR activation using ryanodine (Ry), a classic nonspecific RyRs blocker .To confirm that blocking RyRs does not affect TM-triggered UPR activation, we assessed ectively , A and Bctivator ). As shonockdown , A and B2+ oscillations, and the inclusion of Dan lowered the percentage to 10% (A and B). In addition, we saw increased ratio of spliced XBP1/total XBP1, ATF4, and CHOP mRNA after a 6 h treatment that was not affected by Dan (C–E) or Ry. Although Ry seemed to suppress TM-induced upregulation of XBP1s/XBP1t ratio and CHOP mRNA, the difference was not statistically significant (F and G).We also treated islets with a much lower dose of TM (300 nM) for 24 h and verified if such low dose of TM could trigger UPR activation. We observed that 90% of TM-treated islets exhibited subthreshold Cae to 10% , A and Bd by Dan , C–E or nificant , F and G2+]cyto oscillations (CHOP mRNA levels (A) in INS-1 832/13 cells. We then measured RyR isoforms and IP3R1 mRNA levels in the cells and observed an upregulation of RyR1 mRNA after high glucose culturing, although not significant (p = 0.0832) (B), and unchanged RyR2 (C) or IP3R1 mRNA levels (D). We next tested whether XeC or Dan affected [Ca2+]cyto oscillations (A). Dan significantly reduced the percentage of islets exhibiting oscillations seen following HG treatment from ∼70% to ∼20% (D), and XeC decreased it to ∼30% (average).We previously reported that culturing mouse islets in high glucose  for 16 h induced subthreshold cyto oscillations in TG -treated mouse islets (F), while XeC did not show much affect (I).Thapsigargin (TG) is another widely used ER stress chemical inducer . We preve islets F, while h affect I.Programmed cell death or apoptosis has been shown to occur in beta cells in response to prolonged ER stress , 34, 35.db/db mice, an animal model of type 2 diabetes. These mice harbor a mutation in the leptin receptor and have been shown to exhibit a left shift in their glucose sensitivity over time (db/db mice compared to control mice (db/db mice rapidly gained weight (34.73 ± 4.996 g SD) compared to their heterozygous controls (19.30 ± 4.073 g SD) and developed hyperglycemia , while db/+ mice maintained normal blood sugar . After isolation, islets from the mice were treated with control DMSO or Dan for 16 h before [Ca2+]cyto measurements were carried out. As shown in Figure 11A, islets from db/+ mice treated with vehicle lacked oscillations in 5 mM glucose, and as expected, exhibited normal oscillations in response to 11 mM glucose; these responses were unaffected by pretreatment with Dan. In contrast, db/db mice exhibited oscillations in 5 mM glucose and plateaus in 11 mM glucose. In these islets, the inclusion of Dan abolished the oscillations and decreased the percentage of oscillating islets observed from ∼70% to 25% in 5 mM glucose (B), without affecting the plateaus seen in 11 mM glucose (A).To test the possible relevance of these results to diabetes, we extended our study to islets isolated from ver time  and uprerol mice , 39. We  glucose B, withou glucose A.Figure 2+]cyto oscillations and increased insulin secretion observed in ER-stressed beta cells in subthreshold glucose are triggered by ER Ca2+ reduction and SOCE activation   , 40. How2+]ER in 5 mM glucose, blocking SERCA with CPA further decreased basal [Ca2+]ER (F), but to a lesser degree compared to control (G) as [Ca2+]ER was lower in TM-treated beta cells (B and C). While blocking IP3Rs with XeC also prevented a loss in [Ca2+]ER in TM-treated islets (B), the IP3R blocker XeC failed to inhibit TM-induced subthreshold [Ca2+]cyto oscillations (C) or beta-cell apoptosis (A). On the other hand, blocking RyR1 with Dan suppressed the TM-induced subthreshold [Ca2+]cyto oscillations (D) and beta-cell apoptosis (B) in addition to preventing the reduction in [Ca2+]ER induced by TM (C). Similar results were reported in a study of Dan as a potential treatment for Wolfram syndrome, a rare autosomal recessive disorder that is associated with childhood-onset diabetes mellitus as well as sensory and neurological deficits . Moreover, Dan inclusion  also inhibited TG-induced cell death in neural progenitor cells derived from the induced pluripotent stem cells of a Wolfram syndrome patient , we also exposed islets to glucotoxic  or GLT conditions  to mimic the milieu beta cells are exposed to in T2D; these treatments also trigger ER stress (A) . In addition, we observed that subthreshold [Ca2+]cyto oscillations were prevented by Dan in islets from db/db mice . W. WC), we/db mice . These det al. showed that blocking RyRs with ryanodine prevented TM-induced sub-threshold [Ca2+] oscillations and cell apoptosis and suggested RyR2 to be the mediator due to its abundance in beta cells , in accordance with results we obtained using Dan or ryanodine (D–F and C and D). These data taken collectively suggest that [Ca2+]ER reduction in our system may be downstream of UPR activation by TM, although we are not ruling out other mechanisms, such as reducing SERCA activity (Yamamoto ta cells . As ryanta cells ), we sil did not  and 5. M did not , A and B D–F and , C and Dactivity , 44.2+ depletion to ER stressors nonetheless had differential effects on beta-cell apoptosis and SOCE activation/subthreshold [Ca2+]cyto oscillations seems paradoxical, if ER Ca2+ depletion is indeed causative. However, it may be that the two classes of ER Ca2+ channels have different spatial distributions such that RyRs due to their localization within the cell are capable of selectively activating ER Ca2+ release in the vicinity of the SOCE mechanism. Thus, these receptors may not be uniformly distributed within the cell but selectively and dynamically organized into specific subcellular domains (2+ release-activated Ca2+ channel protein 1 (ORAI1) are the molecular subunits of SOCE channels. STIM1 resides in the ER membrane where it monitors the level of Ca2+ in the ER lumen (2+ entry from the extracellular space into the cytosol (SOCE)  , 41. In than Dan .2+, individual portions of the ER may be too small or poorly sampled by D4ER to resolve differences in these very small and highly localized subdomains. For example, there may be portions of the ER Ca2+ reticular network where RyR1 is dominantly expressed over RyR2 or IP3Rs and which is in closer proximity to SOCE. Therefore, although inhibiting IP3Rs prevented a global ER Ca2+ loss in response to TM, SOCE activation was not inhibited because ER Ca2+ was still low in a STIM1 localized subcompartment of the ER. On the other hand, RyR1, despite its relatively lower abundance in beta-cells, is selectively localized near STIM1 and ORAI1, making the SOCE compartments very sensitive to Ca2+ release by RyR1 localized to this ER Ca2+ pool. Resolving the spatial localization of these molecules within the ER will require higher resolution imaging approaches than that used here, ones that can quantitatively and accurately assay the subcellular localization of RyR1, IP3Rs, STIM1, and ORAI1 under various ER stress conditions.For this scenario to hold, however, we posit that while D4ER indeed measures global ER Ca2+ loss and downstream alterations in beta-cell function and viability. Combining these new data with existing knowledge of RyRs and IP3Rs suggests that RyR1 might be a potentially useful therapeutic target for treatment during the onset or progression of type 2 diabetes.In summary, the present report demonstrates that RyR1 is a critical player in ER stress-induced ER CaTM, CPA, TG, and XeC were obtained from Cayman Chemical, Ann Arbor, MI. Dan was from Sigma-Aldrich, St Louis, MO. Ryanodine (Ry) was from Abcam, Waltham, MA. RNeasy mini kit for RNA extraction was from Qiagen, Germantown, MD. Superscript RT II was from Invitrogen, Carlsbad, CA. SYBR Green PCR master mix was from Applied Biosystems, Bedford, MA. Primers for qRT-PCR were from Integrated DNA Technologies, Coralville, Iowa. Small interfering RNAs (siRNAs) were from ThermoFisher scientific, Waltham, MA. The catalog and lot numbers of these materials can be found in db/db mice (BKS.Cg-Dock7m+/+Leprdb/J) and female heterozygous mice at 5 to 7 weeks of age, as described  and female escribed . Animal escribed .2 humidified atmosphere and used for experiments after reaching ∼70% confluency. INS-1 832/13 cells were transfected with RyR1-specific siRNA, RyR2-specific siRNA, or negative control siRNA using lipofectamine RNAiMAX reagent as described in the manufacturer’s protocol (Invitrogen). Transfection was assessed by qPCR.INS-1 832/13 cells were grown in standard RPMI 1640 medium as described above in 6-well plates at 37 °C in a 5% COTotal RNA was extracted from INS-1 832/13 cells and reverse-transcribed to cDNA as described . qPCR wa2, 5 KCl, 2 MgCl2, 10 HEPES, and 5 glucose. Similarly, INS-1 832/13 cells seeded on glass coverslips were loaded with Fura-2/AM (2.5 μM) for 30 min in standard RPMI 1640 medium containing 11 mM glucose. Coverslips containing INS-1 832/13 cells were transferred to the perfusion chamber and imaged in imaging buffer. Ratiometric Fura-2/AM imaging was performed using 340/380 nm excitation and collecting 502 nm emission, as previously described (Islets were loaded with Fura-2/AM (2.5 μM) for 45 min in RPMI medium including 5 mM glucose. Islets were then transferred to a 1 ml perfusion chamber containing imaging buffer for 6 min, followed by perfusion at ∼1 ml/min. Imaging buffer contained (in mM): 140 NaCl, 3 CaClescribed . Fluores2+]ER was measured using an ER-localized FRET biosensor D4ER . Dz was included to keep the KATP channel in its open state to prevent oscillatory Ca2+ activity and improve the signal/noise ratio and stability of the ER Ca2+ recordings. FRET ratios were acquired using Metafluor software, plotted using Prism, and mean values were calculated using Excel.[Casor D4ER . The samINS-1 832/13 cells were harvested and prepared for sub-G1 apoptosis assay as described . The pert test (Prism) with hypothetical value set for 1.0 or student’s t test when comparing two groups as indicated in the figure legends. Differences between two or more groups were analyzed using ordinary one-way ANOVA or two-way ANOVA (Prism) as specified in the figure legends with post hoc multiple comparisons by Tukey’s procedure. Values of p < 0.05 were considered statistically significant.Data were expressed as means ± SD and analyzed using one sample All data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Five-membered heteroaromatic rings, in particular, have gained prominence in medicinal chemistry as they offer enhanced metabolic stability, solubility and bioavailability, crucial factors in developing effective drugs. The unique physicochemical properties and biological effects of five-membered heterocycles have positioned them as key structural motifs in numerous clinically effective drugs. Hence, the exploration of five-ring heterocycles remains an important research area in medicinal chemistry, with the aim of discovering new therapeutic agents for various diseases. This review addresses the incorporation of heteroatoms such as nitrogen, oxygen and sulfur into the aromatic ring of these heterocyclic compounds, enhancing their polarity and facilitating both aromatic stacking interactions and the formation of hydrogen bonds. Histone deacetylases are present in numerous multiprotein complexes within the epigenetic machinery and play a central role in various cellular processes. They have emerged as important targets for cancer, neurodegenerative diseases and other therapeutic indications. In histone deacetylase inhibitors (HDACi’s), five-ring heterocycles perform various functions as a zinc-binding group, a linker or head group, contributing to binding activity and selective recognition. This review focuses on providing an up-to-date overview of the different five-membered heterocycles utilized in HDACi motifs, highlighting their biological properties. It summarizes relevant publications from the past decade, offering insights into the recent advancements in this field of research. Heterocyclic compounds are a class of organic compounds that contain at least one atom other than carbon in the ring structure. The presence of heteroatoms such as nitrogen, oxygen and sulfur in the aromatic ring increases the polarity and offers the possibility of hydrogen bonds that can improve the binding affinity and selectivity of the drug at the target site. Heteroaromatic rings are considered preferred structures due to their ability to replace common motives in medicinal chemistry, enabling an improvement of metabolic stability, solubility and bioavailability, which are essential elements for the development of effective drugs ,2,3. TheThe structure of typical HDAC inhibitors (HDACis) can be simply illustrated consisting of three parts shown in The aim of this review is to provide an overview of the different five-membered heterocycles in HDACi motifs and their biological properties. In particular, this will summarize publications from the last 10 years to provide an up-to-date overview. HDACis containing five-membered heterocycles are divided into two subchapters according to the localization of the heterocycle scaffold and its interaction with the target enzyme. Inhibitors in chapter 2 contain five-ring heterocycles that bind the catalytic zinc ion in the active site or interact with the foot pocket of certain HDAC isozymes. HDACis in chapter 3 contain five-ring heterocycles integrated either in the linker or in the cap group, allowing diverse molecular interactions with the protein surface.1 (1.2 D) or substituted 2 (1.8 D) azoximes is lower compared to other oxadiazoles  HDACi scaffolds were published by Lobera et al. in 2013 and exhibit outstanding class IIa selectivity TMP195. Th. Th18F] 17 by Hebach et al. [3 group at the C5-position. Replacement with CF2H and a CF3-C3 regioisomer led to a significant decrease in activity. A SAR study of the cap moiety aimed to improve the metabolic stability led to a replacement of the basic amine with a pyrrolidine group and reduced the overall lipophilicity by a factor of 0.6 leading to 18  by a HDAC4 genetic suppression in the R6/2 mouse model  Stott eth et al.   using a MTT assay showed strong synergistic effects for 20 and TMP269 with CI values below 0.5. In two of the four cell lines the synergistic effect of 20 was superior to that of TMP269 with concentrations that retained class IIa preference. Interestingly, 20 is 4–7.7-fold less cytotoxic in leukemia and non-cancer HEK293 cells but still increases caspase activation and apoptosis in a synergistic manner. Compound 20 showed synergistic effects at low concentrations of BTZ while synergistic effects for TMP269 were only observable at elevated concentrations. Another benefit is the induction of p21 in combination with BTZ compared to TMP269 plus BTZ and the stronger caspase-mediated apoptosis. Remarkably, 20 had superior effects compared to TMP269 although both compounds showed similar inhibitory activity in recombinant HDAC enzymes. The authors stated that the general low class IIa HDACi toxicity and more precisely the lower cellular toxicity of 20 together with the synergistic effect could reduce the therapeutic dose of BTZ and result in fewer side effects compared to TMP269 or pan-HDACi.To address the poor 5-year survival of patients with acute myeloid leukemia (AML) and increase treatment success Bollmann et al. were interested in synergistic approaches using a combination of a HDACi and a proteasome inhibitor . Prior selded 20 , which e21 with a tilt of +3.3° and −3.3° for the pyridine rings. 1,3,4-oxadiazole exhibits bond orders of 1.3124, 1.9062 and 1.3348 for the O-C, C-N and N-N bond respectively [2 and 22, concluded that the 1,3,4-oxadiazole is superior to the 1,2,4-motive regarding the pharmaceutical properties [22 regarding the hydrogen bond acceptor strength also showed the 1,3,4-moiety to be a more favorable hydrogen bond acceptor [1,3,4-oxadiazole is a nearly flat and thermally stable heterocycle. It is not fully aromatic, with an index value of 50, compared to furan and thiophen with an index value of 43 and 66, respectively . Bond leectively . A syste23 are known to undergo hydrolysis in basic or acidic conditions and to fragment into carboxylic acids and hydrazine  moiety was reported in 2017 twice in patents by Lee et al.  and Kim 24 and zHDAC6-CD2 resulted in a structure with 1.6 Å resolution (PDB-ID: 8A8Z) and showed no major conformational changes compared to the ligand free structure of zHDAC6-CD2 (PDB-ID: 5EEM). Most interestingly, the electron density in the catalytic site did not match 24 and suggested ring opening of the DFMO moiety. This could be explained by a two-step hydrolytic conversion involving the catalytic activity of HDAC6 with 25 as intermediate, finally yielding a hydrazide as ZBG 26  measurements showed the hydrated form of 24 to be the predominant species to coelute with zHDAC6-CD2 and could exclude complexes of 24 and 26 to be the long-lived complex suggesting the in situ formed acyl hydrazide 25 or the closed hydrated intermediate to be the high-affinity species. Supplementary mutation studies involving the residues Y745 and H574, which are known to be involved in stabilization and orientation as well as deprotonation events were performed. The Y745F mutant showed a faster hydrolysis of 24 with one order of magnitude greater dissociation rate constant indicating involvement in the stabilization of the complex. The H574A mutant exhibited a low hydrolysis rate of 24 and 25 implying the hydrolysis reaction to be enzyme catalyzed and the H574 to be involved. Concurrent studies at the Hansen group led to a similar report by König et al. [27 exhibiting an IC50 value of 193 nM towards HDAC6 and no activity against HDAC1-4  showed a root-mean-square deviation of 0.177 Å indicating no major structural changes. Remarkably, and in agreement with data of Cellupica et al., the DFMO moiety did not match the found electron density thus suggesting a ring opening reaction based on DFMO reactivity. Interestingly and supplementary to data of Cellupica et al., the found electron density matched acylhydrazine 28, the ring opening product perfectly. The proposed mechanism includes an attack of zinc bound water on the DFMO C2 carbon yielding the ring opened acylhydrazide 28 which is stabilized by many intermolecular interactions. The key interaction is a coordination of the zinc ion by the nitrogen anion with a distance of 2 Å. Further interactions include hydrogen bonds between H574 as well as G743 and a carbonyl group of the acylhydrazine moiety. Additional hydrogen bonds are present between the amide carbonyl and Y745 and the difluoro group and C584 and Y745. A comparison with the structure published by Cellupila et al. showed a shift in orientation of the ZBG by 0.9 Å and a coordination of the zinc ion by the primary amine of the hydrazide moiety. Further differences include a shorter hydrogen bond towards Y745 and the presence of water in the P571 pocket. To shed more light on the binding mechanism the acylhydrazine 28, the TFMO 29, and the methyl moiety 30 were synthesized and evaluated. Compounds 28 and 29 showed only weak inhibition of HDAC6 whereas 29 was more potent with an IC50 of 531 nM. Further evaluation of binding kinetics was performed with 27 and 29 showing incubation time dependent inhibitory activity . Jump dilution experiments with 27 showed very slow off kinetics which could be confirmed by dialysis experiments indicating a near irreversible inhibition of HDAC6. In contrast, 29 exhibited fast-off kinetics validated in jump dilution experiments, which could be rationalized by a difference in binding mode induced by the sterically more demanding trifluoro group, leading to clashes, thereby destabilizing the binding in the P571 pocket. Further elucidation of the slow binding mechanism suggested “simple slow-binding” mechanism I for 29 and “induced fit” mechanism II for 27. Essentially, 27 is a slow on inhibitor which serves as substrate analogue with a near irreversible binding mode upon conversion in the active site.Modelling suggested an attack of activated water at the DFMO moiety leading to a hydrated intermediate able to engage in hydrogen bonding (HB) with two Histidine residues (His573 and His574) and to preserve interactions with Tyr745. This intermediate could further undergo ring opening and yield an acyl-hydrazide moiety which in turn could serve as substrate for HDAC6 or be hydrolyzed in solution. Conclusively, the hydrazide group would engage in monodentate binding and simultaneously act as a HB donor towards catalytic key residues His573 and His574, whereas the carbonyl oxygen would engage in interactions with Tyr745, acting as HB acceptor. The inherent selectivity towards HDAC6-CD2 was also explained by modelling, suggesting unfavorable geometry in other HDAC isozymes and HDAC6-CD1 for the first hydrolysis step. Further studies of the binding properties revealed a slow binding with an association rate constant of 9.6 × 10 potency  and seleg et al.  confirmi HDAC1-4 . Additio31 and 32  and developed the first proteolysis-targeting chimeras (PROTACs) with the selective, non-hydroxamate DFMO moiety . Inspire1 and 32  were sel33 (33 exhibited time dependent inhibitory activity (4.6 nM–60 min) but was highly potent even without preincubation (36 nM). Due to the high selectivity of the DFMO moiety concentrations of up to 10 µM had no obvious impact on HDAC1, 4 or 7. Examination in primary neural mice cells showed concentration dependent acetylation of tubulin without affecting H3 acetylation. In vivo experiments showed that oral administration of 33 led to an increase of tubulin acetylation and plateaued at 1–4 h, gradually returning to control levels after 24 h with values of 1037 ng/mL, 0.25 h, 1722.5 ng·h/mL and 1.76 h for Cmax, Tmax, AUC0–24 h and MRT, respectively. The effect of HDAC6 inhibition was validated using wild type (WT) and HDAC6 KO mice, showing elevated tubulin acetylation in WT but not KO mice. Therapeutic benefits were estimated by administration of 33 for 2 weeks using the P301S tau Tg tauopathy mouse model. A dose of 3 mg/kg in 5-months old mice significantly improved axonal function. The same dose significantly decreased age-dependent tau accumulation in 6- to 9-months-old mice in the FA fraction without affecting the RAB-soluble and RIPA-soluble fractions and led to behavioral improvement regarding the novel object recognition deficit. The authors concluded that 33 can benefit axonal function but that the underlying mechanism of action has not been fully elucidated in part because of HDAC6 versatility and the involvement in various biological pathways.In search of a therapeutic strategy for tauopathy, Onishi et al. synthesized and evaluated compound 33  via HTS 34  are five-membered non-aromatic heterocycles which can structurally be derived from thiazole, its aromatic analogue. The TZD moiety can act as a hydrogen bond donor/acceptor and shows no significant deviation from planarity with bond angles and distances indicated in Thiazolidinediones (TZDs) are mainly known for their role in type 2 diabetes, acting as PPARγ agonists thereby decreasing insulin resistance. Elucidation of side effects led to a decline in clinical use and a drop in research publications but the versatile nature of this scaffold promotes the application in many areas including anti-fungal, anti-bacterial and anti-cancer agents and as zinc binding groups ,46.35, 36 and 37  resulted in more favorable binding energies than docking into the closed conformation of HDAC4c (PDB-ID: 4CBY). Interactions of the TZD moiety in the active site of HDAC4 composed of complexation of the zinc ion via the C4 carbonyl, a hydrogen bond with His159 via the amide nitrogen and a π-sulphur interaction with Phe168. All tested compounds exhibited a wide range of activity, of which most were two-fold more active on HDAC4, a few were dual inhibitors of HDAC4 and HDAC8 and one compound was selective for HDAC8 but exhibited poor potency. Further optimization identified the benzothiazole moiety as the best cap group and 38 as the most potent compound  and solid tumor (MDA-MB-231) cells with low impact on non-cancerous HS-27 cells.Subsequent efforts via a permutation approach yielded a series of overall more elongated compounds with significant increase in activity which were shown to bind to HDAC4 via the TZD scaffold in in silico studies . Dockingcompound  towards o (PDB-ID: 2VQJ) with the enlarged pocket resulted in more favorable binding energies compared to closed HDAC4. Docking in HDAC8 showed similar results to prior studies indicating complexation of the zinc ion via the amide carboxyl group. The most potent compound 40  indicated an interaction of the TZD moiety with zinc via the C2 carbonyl, differing to previous docking results suggesting a complexation via the C4 carbonyl. No significant difference was observed upon docking the enantiomer. Evaluation in cancer cell lines MCF-7, K562, A549 and HT-29 showed IC50 values of 28.14 µM, 46.27 µM, 19.52 µM and 18.84 µM, respectively. In addition, 42 inhibited in vitro HUVEC  proliferation (IC50 < 10 µM), migration and tube formation and repressed the formation of new capillary in an in vivo CAM assay.Similar studies were performed by Upadhyay et al.  targetin42  was most43  showed higher docking scores for the open conformation and higher binding scores for a (S)-enantiomer analogue. Again, the TZD moiety binds the zinc ion with the C4 carbonyl, forms a hydrogen bond to H159 with its amide moiety and is engaged in π-π interactions with H198. Further evaluation of 43 showed an IC50 of 2 µM against HUVECs and IC50 values of 16.92 µM, 8.92 µM, 17.99 µM and 8.20 µM against MCF-7, K562, A549 and HT-29 cell lines, respectively. Additionally, 43 inhibited endothelial cell proliferation, migration, tube formation and suppressed new capillary formation in growing chick chorioallantois membranes (CAMs). In vivo evaluation in mice xenograft models of human colorectal adenocarcinoma (HT-29) showed a regression in tumor volume by administration of 43 (25 mg/kg) comparable to the reference doxorubicin (20 mg/kg).Further studies Upadhyay et al.  led to a43  was most50 values under 50 µM which were used for further SAR analysis. A similarity mapping with the DataWarrior software yielded ten clusters , HCT116 (3.21 µM), SW620 (4.43 µM), Lovo (5.03 µM) and MCF-7 (7.12 µM) [58 and 59 were evaluated against eight cancer cell lines with following IC50 values HCT-116 (1.09 µM and 2.73 µM), U937 (12.36 µM and 11.27 µM), B16 (4.97 µM and 30.48 µM), PC-3 (9.18 µM and 14.20 µM), HeLa (9.18 µM and 20.25 µM), HEL (1.26 µM and 3.14 µM), KG1 (0.37 µM and 6.69 µM) and ES-2 (5.07 µM and 44.39 µM), respectively [In an effort to design nitric oxide (NO)-donating scaffolds Tu et al.  and Duan8 and 59  were mossylation ,62. EvalEntinostat [SAHA [Entinostat [61 and 62 , A549 (6.39 µM and >100 µM), NCI-H661 (4.73 µM and >100 µM), U937 (0.52 µM and 7.21 µM) and MDA-MB-231 (3.18 µM and 14.67 µM). Studies on SAHA [63, 64 and 65 , K562 (4.15 µM and 1.56 µM) and PC3 (7.75 µM and 3.60 µM) cancer cell lines, respectively. On the basis of previous results Yang et al. [68 and 69, which performed better than SAHA against HDAC1. Evaluation against cancer cell lines yielded following IC50 values: HCCLM3 (5.19 µM and 6.56 µM) and HepG2 (1.07 µM and 1.03 µM) for 68 and 69, respectively. Pidugu et al. [70 and evaluated it in a follow up study [50 values of 0.23 µM and 1 µM, respectively. The compound 71, synthesized and evaluated by Yang et al. [Several other approaches including substitution and optimization strategies yielded compounds The triazole scaffold is widely applied and popular as the preparation via the copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) fulfills all criteria for a click reaction: being simple to perform, wide in scope, stereospecific and highly yielding as well as modular with easy separatable non-toxic byproducts. Many research groups applied the CuAAC reaction in the synthesis of compound libraries or as part of optimization strategies which will be presented in the following.72  and PC3 (1.4 µM and 1.0 µM) cell lines, respectively.In 2010 Suzuki et al.  identifi72  as part 72 , PC3 (7.98 µM and 2.62 µM), AsPC-1 (4.18 µM and 1.39 µM) and NCI-H23 (5.46 µM and 1.14 µM) for 78 and 79, respectively. Follow up studies by Dung et al. [80 and 81 , PC3 (0.76 µM and 1.74 µM) and AsPC-1 (0.49 µM and 1.49 µM). Further screening and improvements by Dung et al. [82 and 83 , PC3 (2.17 µM and 2.24 µM), AsPC1-1 (3.03 µM and 1.48 µM) and NCI-H23 (2.89 µM and 0.83 µM).In search for novel HDACi Huong et al.  used the8 and 79  were mosg et al.  yielded 0 and 81 , with po2 and 83 . Evaluat84 and 85  of representative compounds 86 and 87 (el (0.28·h−1 and·0.14·h−1), t1/2 (2.49 h and 5.03 h), tmax (0.42 h and 0.25 h), Cmax (9558 ng·mL−1 and 2715 ng·mL−1) and an AUC0–t (15278 h·ng·mL−1 and 2917 h·ng·mL−1), respectively with an oral bioavailability of 65.1% for 86. Evaluation in several cancer cell lines showed acetylation of histone H3 and histone H4 in a concentration-dependent manner and a G0/G1 cell cycle arrest with caspase-dependent apoptosis. Antitumor evaluation of 86 in the MC38 homograft and HCT116 xenografted athymic nude mice inhibited tumor growth and elevated CD4+ T cell levels in MC38 homograft. Combinatorial treatment in MC38 mice with 86 and mPD-1 antibody, an immune checkpoint inhibitor led to tumor growth inhibition and complete tumor regression in 5 out of 6 mice, without obvious side effects.In an effort to improve antitumor activity and reduce adverse side effects Sun et al. designed and synthesized selective and potent HDAC1 inhibitors incorporating a triazole isomer . An SAR 6 and 87  yielded 88 of series II was not as potent compared to series I compounds but exhibited better antiproliferative activities presumably acting as tubulin inhibitor.In search of novel HDACi, Aboeldahab et al.  synthesi72 , RPMI (0.085 µM), NCI-H929 (1.23 µM), LP1 (0.37 µM), Mino (0.12 µM) and JeKo-1 (0.064 µM) [90  . Compoun µM) [90 , equippebinostat  as contr91, which displayed high cytotoxicity in several cancer cell with IC50 values better or comparable to SAHA and exerted potent antitumor effects in the A-549 zebrafish xenograft model.Further studies incorporating the thiazole motive were conducted by Sun et al.  yielding92 which was synthesized by Nam et al. [Another example is the HDAC6 selective compound m et al.  based onPlasmodium falciparum Bresciani et al. [93O , MCF-7 (1.81 µM), BGC823 (0.26 µM), A549 (0.26 µM), HT1080 (1.33 µM), K562 (0.46 µM), U973 (0.51 µM) and Molt-4 (0.17 µM). [The pyrazole scaffold was used in several publications as part of the cap group (Compounds than 109 . A SAR s.17 µM). 111 as most potent compound , HT-29 (10.20 µM and 6.92 µM), MCF-7 (12.23 µM and 7.15 µM), MDA-MB-231 (>50 µM and 27.31 µM), A549 (14.43 µM and 6.07 µM), PC-3 (11.03 µM and 4.47 µM) and AsPC-1 (38.85 µM and 25.31 µM), respectively [Subsequent studies by Wen et al.  further 112  against ectively .114 being the most selective for HDAC6  piperidine showed good activity against HDAC1 and HDAC6 . Compounds with piperazine linker, 118 showed comparable values against HDAC6 . Also, good antiproliferative activity like cell cycle arrest and apoptosis of human cancer cell lines, tested with pro-monocytic human myeloid leukemia (U937) cells and triple-negative breast cancer (MDA-MB-231) cells, could be achieved [N-bis-substituted aromatic amide hydroxamic acid derivatives. Thereby IC50 of 1.14 nM for HDAC1 was achieved for one compound, 119 . IC50 values for the WT was much higher with 367 nM for TFMK and 978 nM for HA [Thiophenes can be utilized as capping groups in HDAC inhibitors. For example, tetrahydrobenzo[achieved . Thiopheund, 119 . This mound, 119 . The hydM for HA . Heterocyclic rings, particularly those with five members, are highly valued in the development of active pharmaceutical ingredients. These rings belong to a class of organic compounds that contain at least one non-carbon atom within their structure. The inclusion of heteroatoms such as nitrogen, oxygen and sulfur in the aromatic ring enhances the polarity of the compound and introduces the potential for multiple target-inhibitor interactions. This, in turn, improves the drug’s binding affinity and selectivity at the intended target site. Heteroaromatic rings are considered favorable structures in medicinal chemistry as they can replace common motifs, leading to enhanced metabolic stability, solubility and bioavailability. This review discusses the importance of five-ring heterocycles in HDACis. They occur in three different structural elements of typical HDACis. Five-ring heterocycles can act as ZBG, provide the correct geometry in the binding pocket in the linker part often forming additional hydrophobic or cation-Pi interactions or interact with the protein surface in the head group, which enables the modulation of isozyme selectivity. Of particular interest are five-ring heterocycles that can replace the most commonly used ZBGs in HDACis, such as hydroxamic acid or TFMK. This is significant because these groups potentially mediate non-specific metalloenzyme binding thus increasing the risk for undesired side effects. In addition, hydroxamic acids are suspected of having mutagenic effects and TFMKs are metabolized very rapidly. Five-ring heterocycles can not only replace ZBGs, but also add entirely new beneficial mechanistic properties. The special molecular properties of DFMKs, for example, allows specific cleavage of the oxadiazole ring, presumably by the catalytic machinery of HDAC6, which leads to highly selective HDAC6is with very long residence times. The knowledge on five-membered heterocycles in HDACis summarized here opens up numerous interesting starting points for the future to develop new non-hydroxamic HDACis and provides guidance for medicinal chemists to design and optimize HDACis with improved pharmacokinetic and pharmacodynamic properties.


S100A6 and murine double minute 2 (MDM2) are important cancer-related molecules. A previous study identified an interaction between S100A6 and MDM2 by size exclusion chromatography and surface plasmon resonance experiments. The present study investigated whether S100A6 could bind to MDM2 in vivo and further explored its functional implication.S-transferase pull-down assay, and immunofluorescence were performed to determine the in vivo interaction between S100A6 and MDM2. Cycloheximide pulse-chase assay and ubiquitination assay were performed to clarify the mechanism by which S100A6 downregulated MDM2. In addition, clonogenic assay, WST-1 assay, and flow cytometry of apoptosis and the cell cycle were performed and a xenograft model was established to evaluate the effects of the S100A6/MDM2 interaction on growth and paclitaxel-induced chemosensitivity of breast cancer. The expressions of S100A6 and MDM2 in patients with invasive breast cancer were analyzed by immunohistochemistry. In addition, the correlation between the expression of S100A6 and the response to neoadjuvant chemotherapy was statistically analyzed.Co-immunoprecipitation, glutathione-S100A6 promoted the MDM2 translocation from nucleus to cytoplasm, in which the S100A6 bound to the binding site of the herpesvirus-associated ubiquitin-specific protease (HAUSP) in MDM2, disrupted the MDM2–HAUSP–DAXX interactions, and induced the MDM2 self-ubiquitination and degradation. Furthermore, the S100A6-mediated MDM2 degradation suppressed the growth of breast cancer and enhanced its sensitivity to paclitaxel both in vitro and in vivo. For patients with invasive breast cancer who received epirubicin and cyclophosphamide followed by docetaxel (EC-T), expressions of S100A6 and MDM2 were negatively correlated, and high expression of S100A6 suggested a higher rate of pathologic complete response (pCR). Univariate and multivariate analyses showed that the high expression of S100A6 was an independent predictor of pCR.These results reveal a novel function for S100A6 in downregulating MDM2, which directly enhances sensitivity to chemotherapy.The online version contains supplementary material available at 10.1186/s13058-023-01657-w. As a multifunctional oncoprotein, murine double minute 2 (MDM2) is found to be overexpressed in patients with a variety of cancers , includiExpression of MDM2 can be regulated through multiple pathways, such as gene amplification, p53-mediated transcriptional induction, and post-translational regulation by self-ubiquitination . MDM2 is2+-binding protein that belongs to the S100 protein family [2+-binding domains in each monomer [2+ will induce a conformational change in the S100A6 protein to expose the hydrophobic amino acid residues to interact with the target proteins [2+ homeostasis, protein degradation control, and transcription factor regulation, thereby affecting cell proliferation, differentiation, apoptosis, stress response, and many other biological events [S100A6  is a Can family . Similar monomer . Bindingproteins . There aproteins –18. Thesl events .S-transferase (GST) pull-down assay, and immunofluorescence. Further studies found that S100A6 bound to the binding site of the deubiquitinating enzyme HAUSP in MDM2, disrupted the MDM2–HAUSP–DAXX interactions, and induced MDM2 self-ubiquitination and degradation. Moreover, the S100A6-mediated MDM2 degradation suppressed the growth of breast cancer and enhanced sensitivity to paclitaxel both in vitro and in vivo. For patients with invasive breast cancer who received epirubicin and cyclophosphamide followed by docetaxel (EC-T), the expressions of S100A6 and MDM2 were negatively correlated, and S100A6 was a predictive biomarker for the efficacy of neoadjuvant chemotherapy. This study proposes a novel and important function of S100A6 in regulating MDM2 that directly affects the growth of tumor cells and their sensitivity to chemotherapy, which is of high value in clinical use.MDM2 has been identified as another target protein of S100A6. Size exclusion chromatography and surface plasmon resonance experiments show that S100A6 binds directly to MDM2 in vitro . HoweverIn this study, six breast cancer cell lines  were selected and supplied by the American Type Culture Collection (ATCC) and then cultured as instructed. In addition, paclitaxel was purchased from Sigma-Aldrich, and the stock solution was diluted in dimethyl sulfoxide (DMSO) at 4 mM. Cycloheximide  and MG132 (10 mM) were purchased from MCE and were diluted in DMSO before use.Various truncated or mutated human S100A6, MDM2, and HAUSP constructs were generated by using polymerase chain reaction (PCR) and then cloned into pCMV-Flag, pCMV-Myc, or pCMV-HA expression vectors (Clontech). Meanwhile, various truncated human S100A6 and MDM2 constructs were generated by PCR and cloned into T7 promotor-GST or T7 promotor-HA expression vectors (Clontech). MDM2 mutations included MDM2 C461S (substitution of Cys by Ser), MDM2 C464A (substitution of Cys by Ala), and MDM2 C478S (substitution of Cys by Ser). The cells were transfected with S100A6 siRNA (CCUCUCUGAGUCAAAUCCATT) with Lipofectamine 3000 (Invitrogen). The overexpression rate and knockdown effect were detected by western blot.The expressions of S100A6, MDM2, HAUSP, DAXX, Ubiquitin, HA, GST, Flag, and Myc proteins were measured by western blot. The employed antibodies were listed as follows. S100A6 antibody (ab 181975), HAUSP antibody (ab 264422), HA tag antibody (ab 9110), GST-tag antibody (ab 138491), and Flag tag antibody (ab 205606) were purchased from Abcam. MDM2 antibodies were purchased from Abcam (ab 259265), Cell Signaling Technology (D1V2Z), and ABclonal (A0345). Ubiquitin antibody was purchased from ABclonal (A19686). Myc tag antibodies were purchased from Abcam (ab 32972) and Santa Cruz (sc-40). DAXX antibody was purchased from Santa Cruz (sc-8043). The concentrations of all antibodies were applied strictly according to the manufacturers’ instructions.S100A6 and MDM2 plasmids were co-transfected into MCF-7 cells. Twenty-four hour after transfection, cells were lysed in ice-cold immunoprecipitation lysis/wash buffer . The single colonies were selected and cultured in ampicillin-resistant liquid LB medium, shocked at 37 °C until the optical density reached 0.5, and then shocked again at 20–23 °C overnight with the addition of IPTG (0.5 mmol/L). After the recombinant proteins were obtained, HA-MDM2 or its fragments (carried His-tag) and GST-S100A6 or its fragments (carried GST-tag) were purified with a purification kit  separately according to the instructions.Approximately 100 µg of mixture composed of GST or GST-S100A6 or its fragments and HA-MDM2 or its fragments were immobilized in 100 µL of glutathione agarose (Biolinkedin) for incubation overnight at 4 °C under gentle rotation. The magnetic beads were retained, rinsed, and eluted in 1 × SDS loading buffer. Finally, the western blot was performed on the bound proteins.MCF-7 cells were transfected with Myc-MDM2 and/or Flag-S100A6 plasmids for 24 h. They were rinsed with phosphate buffer saline (PBS), fixed with pre-chilled 4% paraformaldehyde for a quarter hour, and then permeabilized with 0.1% Triton X-100 for another quarter hour at ambient temperature. Next, they were blocked with 5% BSA for 60 min at the same temperature as above and then incubated with an anti-Myc rabbit antibody  and anti-Flag mouse antibody  at 4 °C overnight. After that, they were rinsed with PBS (3 times × 5 min) and stained with Fluorescein (FITC)-conjugated AffiniPure Donkey Anti-Rabbit IgG  and Rhodamine Red-X-conjugated AffiniPure Donkey Anti-Mouse IgG  for about 60 min in the dark at room temperature. Again, they were rinsed, 3 times × 5 min, with PBS and then stained with diamamidine phenylindole (DAPI) for 15 min at 37 °C. Images were captured with a laser confocal microscope .Protein turnover of MDM2 was assessed by CHX pulse-chase assay. MCF-7 cells were co-transfected with MDM2 and S100A6/Flag-S100A6 or HA-HAUSP plasmids for 24 h. After that, the protein synthesis inhibitor CHX  was added 15, 30, 60, and 90 min before cell lysis. After CHX treatment, the cell lysates were prepared and the expression of MDM2 was detected by western blot.Cells were treated with the proteasome inhibitor MG132  for 10 h before collection. Cell lysates, after low-temperature centrifugation, were then incubated with A/G magnetic beads and anti-MDM2 antibody on a rotator at low temperature overnight. The beads were washed, and the ubiquitinated products were eluted and detected by western blot using an anti-ubiquitin antibody.Full-length GST-S100A6 and HA-MDM2 and its fragments were expressed and purified as previously described in the GST pull-down assay. The in vitro ubiquitination assay was performed in a total reaction volume (approximately 100μL) consisting of 5 μg E1 (0.5 μg/μL), 5 μg UbcH5 (1 μg/μL), 50 μg HA-MDM2, 10 μg ubiquitin (1 μg/μL), 50 μg GST-S100A6, and 5 μg 10 × Buffer (1 μg/μL). E1, UbcH5, ubiquitin, and 10 × Buffer were purchased from Enzo Life Science. The in vitro reactions were carried out at 37 °C and 380 rpm for 60–90 min, followed by a western blot assay.S100A6, MDM2, or MDM2 and S100A6-transfected MCF-7 cells were prepared in a single-cell suspension. After that, 150 cells were seeded into a 6-well plate, which lasted for about 14 days so that colonies were generated. Paclitaxel was then added to the cell culture medium at various concentrations. After 48 h, the cells, with or without paclitaxel, were rinsed with PBS and then fixed with 4% paraformaldehyde for 30 min and stained with 0.5% crystal violet for about 20 min. After the dying solutions were removed carefully, the colonies generated previously were rinsed with PBS, counted, and calculated.The water-soluble tetrazolium (WST) salt assay was employed in this study to analyze the effect of S100A6 on paclitaxel-induced cytotoxicity. Cells transfected with S100A6, MDM2, or MDM2 and S100A6 were placed in 96-well plates for culture and then treated with paclitaxel at various concentrations for a day and a night (24 h) at 37 °C. The mixture of cells and WST-1 (25 μg/well) was incubated for another 4 h. At this time, the optical density was detected at 450 nm by using 620 nm as a reference.Annexin-V staining followed by flow cytometry was employed to demonstrate the effects of paclitaxel and S100A6 on apoptosis. Cells with S100A6, MDM2, or MDM2 and S100A6 transfection and paclitaxel (100 μM for different times) treatment were harvested, rinsed twice with cold PBS, stained with FITC-Annexin-V and PI (BD Pharmingen) following the manufacture’s suggestions, and then detected by flow cytometry.The cells were treated the same as above and then fixed in 70% ethanol for over 12 h. Then, they were centrifuged and rinsed with PBS twice. Finally, cells were dissolved in 200 μL of PI/RNase Staining Buffer (BD Pharmingen) and detected by flow cytometry to measure the DNA content.6 cells in 150 μL PBS) were subcutaneously inoculated into the right posterior flank of female SPF nude mice. Ten days later, the mice were treated with or without intraperitoneal injection of paclitaxel . After treatment, the mice were humanely killed on day 38, and tumor tissues were used for western blot.Specific pathogen-free (SPF) nude mice  were randomly allocated to 8 groups. MCF-7 cells were infected with lentivirus expressing S100A6, MDM2, or MDM2 and S100A6. The stable transfected cells . They were processed and analyzed with ImageJ, GraphPad Prism, and SPSS, respectively. The normality of data distributions was subjected to the Shapiro–Wilk test. The independent sample T test was employed for comparison between two groups, while one-way or multi-factor analysis of variance was selected for comparisons of three or more groups. Immunofluorescence was analyzed with the Pearson’s correlation coefficient (PCC). Data from clinical samples were analyzed with the Chi-square test and Fisher’s exact test. *To determine whether the S100A6 was correlated with MDM2, six human breast cancer cell lines  were tested firstly to measure the expressions of S100A6 and MDM2. Western blot assay showed that a high expression of S100A6 was closely associated with a low expression of MDM2. MDA-MB-231 and HCC1937 expressed a high expression of S100A6 and low expression of MDM2; SUM159, MCF-7, ZR-75-1, and MDA-MB-468 showed a low expression of S100A6 and relatively high expression of MDM2 , and completely inhibited by Ca2+ chelator ethylene glycol tetraacetic acid (EGTA)  and contains several conserved functional domains . The N-t2+-binding domains [S100A6 usually forms dimers, and each monomer contains two EF-hand Ca domains . Two difIn addition, the subcellular localizations of S100A6 and MDM2 were observed by immunofluorescence experiment. Myc-MDM2, as indicated by red fluorescence, was mainly expressed in the nucleus, while Flag-S100A6, as marked by green fluorescence, was predominantly localized in the cytoplasm. Flag-S100A6 transfection promoted the translocation of Myc-MDM2 from the nucleus to the cytoplasm, where interacted with Flag-S100A6. Digital merging of confocal microscopic images of Myc-MDM2 (red fluorescence) and Flag-S100A6 (green fluorescence) depicted considerable colocalization (yellow fluorescence) of these proteins in the cytoplasm (with a PCC of 0.76) , the MDM2 fragments 1–299, or 300–491 tagged with Myc. Enforced expression of S100A6 increased the ubiquitination of full-length MDM2 (1–491) and MDM2 fragment 300–491. However, S100A6 did not increase the ubiquitination of MDM2 1–299   and MDM2 fragment 300–491, but not MDM2 1–299, underwent ubiquitination by GST-S100A6 in vitro  and MDM2 fragments 1–299 and 300–491, were expressed in To further confirm that S100A6 induced the self-ubiquitination of MDM2, the RING finger mutated MDM2 C461S, C464A, and C478S, which lose E3 activity, were constructed here  but to the 300–436 fragment containing the binding site of the deubiquitinating enzyme HAUSP   . Among them, 55 cases (51.4%) showed high immunoreactivity for S100A6, while 52 cases (48.6%) expressed low immunoreactivity. The Chi-square test revealed that S100A6 was negatively correlated with expression of MDM2 (P < 0.0001)   was investigated. It was found that pCR in patients treated with preoperative chemotherapy (EC-T) was significantly correlated with the expression of S100A6. Specifically, patients with highly expressed S100A6 possessed a significantly higher rate of pCR (1) Table . Univariy) Table .Table 2CIn the present study, it was found that S100A6 bound to MDM2 and exerted an induction effect on MDM2 self-ubiquitination and degradation. It was also found that by disrupting the MDM2–HAUSP–DAXX complex, S100A6 induced MDM2 self-ubiquitination. Meanwhile, S100A6 also recruited MDM2 from the nucleus to the cytoplasm Fig. . ImportaThe co-immunoprecipitation first demonstrated that S100A6 and MDM2 could interact with each other in breast cancer cells. A further observation from the GST pull-down assay showed that S100A6 specifically bound to a fragment (300–436) close to the C-terminal RING domain of MDM2, which was not in line with the finding of a previous study using size exclusion chromatography and surface plasmon resonance. This study showed that S100A6 bound to the N-terminal domain of MDM2 . It was MDM2 is not stable so it can be ubiquitinated and degraded by autocatalysis . HoweverIn addition, overexpressed S100A6 promoted MDM2 shuttling from the nucleus to the cytoplasm and increased its colocalization with S100A6, although the mechanism remains unknown. S100A6 is predominantly located in the cytoplasm , so S100S100A6 is expressed in various cell types of human normal tissues , 31. HowNeoadjuvant chemotherapy promotes the disease-free survival and overall survival rates in combined therapy for breast cancer patients. Meanwhile, it enables more patients to undergo the breast-conserving surgery and significantly improves their quality of life without elevating the rate of local recurrence . As a weIn conclusion, this study demonstrates a novel and important role of S100A6 in the negative regulation of MDM2. By disrupting the MDM2–HAUSP–DAXX-HAUSP-DAXX complex, S100A6 induces MDM2 self-ubiquitination and its degradation, which suppresses the growth of breast cancer and enhances its sensitivity to chemotherapy. These results suggest that S100A6 can be a predictive marker for chemotherapy response and that it may be a significant means to chemotherapy by elevating the expression level of S100A6 in patients with highly expressed MDM2 (Additional file. Additional file 1. Raw data for western blot and colony formation assay.


Fusarium wilt of banana, especially Tropical Race 4 (TR4) is a major factor restricting banana production. Developing a resistant cultivar and inducing plant defenses by elicitor application are currently two of the best options to control this disease. Isotianil is a monocarboxylic acid amide that has been used as a fungicide to control rice blast and could potentially induce systemic acquired resistance in plants. To determine the control effect of elicitor isotianil on TR4 in different resistant cultivars, a greenhouse pot experiment was conducted and its results showed that isotianil could significantly alleviate the symptoms of TR4, provide enhanced disease control on the cultivars ‘Baxi’ and ‘Yunjiao No.1’ with control effect 50.14% and 56.14%, respectively. We compared the infection processes in ‘Baxi’ (susceptible cultivars) and ‘Yunjiao No.1’ (resistant cultivars) two cultivars inoculated with pathogen TR4. The results showed that TR4 hyphae could rapidly penetrate the cortex into the root vascular bundle for colonization, and the colonization capacity in ‘Baxi’ was significantly higher than that in ‘Yunjiao No.1’. The accumulation of a large number of starch grains was observed in corms cells, and further analysis showed that the starch content in ‘Yunjiao No. 1’ as resistant cultivar was significantly higher than that in ‘Baxi’ as susceptible cultivar, and isotianil application could significantly increase the starch content in ‘Baxi’. Besides, a mass of tyloses were observed in the roots and corms and these tyloses increased after application with isotianil. Furthermore, the total starch and tyloses contents and the control effect in the corms of ‘Yunjiao No.1’ was higher than that in the ‘Baxi’. Moreover, the expression levels of key genes for plant resistance induction and starch synthesis were analyzed, and the results suggested that these genes were significantly upregulated at different time points after the application of isotianil. These results suggest that there are significant differences between cultivars in response to TR4 invasion and plant reactions with respect to starch accumulation, tyloses formation and the expression of plant resistance induction and starch synthesis related genes. Results also indicate that isotianil application may contribute to disease control by inducing host plant defense against TR4 infection and could be potentially used together with resistant cultivar as integrated approach to manage this destructive disease. Further research under field conditions should be included in the next phases of study. On the  4 (TR4) . In the  4 (TR4) . In the  4 (TR4) . In the  4 (TR4) . Then it 4 (TR4) . In rece 4 (TR4) , Lebanon 4 (TR4) , Israel  4 (TR4) , Mozambi 4 (TR4) , Pakista 4 (TR4) , Puerto  4 (TR4) , Miyako  4 (TR4) , India ( 4 (TR4) , Mayotte 4 (TR4) , Colombi 4 (TR4)  and Peru 4 (TR4) . As TR4  4 (TR4) , it is e 4 (TR4) , no compIn the natural environment, in order to prevent pathogenic infection, plants not only form a physical barrier on the surface, but also have various internal immune responses. Plants are able to induce broad defense reactions by pathogens in their surroundings . So, actPR) genes , ACS [1-aminocyclopropane-1-carboxylic acid (ACC) synthase] and ACO (ACC oxidase), and ACS is a key synthetase gen in this pathway (ACS) and signaling  is usually upregulated (bHLH) transcription factor (TF) MYC2 as a major regulator in the JA pathway, coordinates plant resistance to pathogens through JA-mediated defense responses  and induced systemic resistance (ISR) . SAR, whR) genes . PR protpathogen . NPR1, a pathway , and bot pathway . In cont pathway . Althoug pathway . ET is s pathway . In geneegulated . The basesponses . In addiesponses .AGPase), Starch branching enzyme (SBE) and granule-binding starch synthase (GBSS) are key enzymes in the starch biosynthesis pathway, and AGPase plays an important role in crop heat tolerance  mimic, with proven activity against rice blast  and whea pathway . There i pathway , and the pathway Figure 122.1Musa spp. AAA, Susceptibility cultivar) and ‘Yunjiao No.1’ , and the banana plantlets were propagated by plant tissue culture. The tissue plantlets were cultured at 25°C, under a 16 h/18 h (light/dark) photoperiod until new roots grew and then transplanted into an aperture disk , filled with coconut bran and seedling substrate. Each banana plantlets about 15 cm high with 5 leaves were transplanted into the plastic pot with 25 cm in the diameter containing garden soil and substrate. All banana plants were grown in a solar greenhouse with isotianil watering and fertilization management.In this study, two cultivars of Cavendish were used, ‘Baxi’  containing 0.2 g/mL isotianil and was applied when the banana plants had 6-7 leaves. The applied diluent was prepared by dissolving 0.035 mL of the original product in 100 mL of tap water per banana plant, applied by either drenching the roots or spraying the leaves . The applications were performed once every 28 days, and the applications were performed three times in total. Seven days after the second application of Routine®, 100 mL of 1×107 spores/mL of TR4 was drenched for root inoculation, and the control treatment was applied with tap water. Before TR4 inoculation, the roots were treated with two wounds around plants, using a shovel. Banana roots and corms were taken 0 d, 1 d, 7 d, 14 d, and 62 d after TR4 inoculation as samples for subsequent TR4 content detection, microscopic observation and gene expression determination.TR4 labeled with green fluorescent protein (GFP) was used to explore the infestation process of pathogens in plants . After T2.3® and inoculated with TR4 (TR4+R1) and Banana root drenched with Routine® and inoculated with TR4 (TR4+R2) (Four treatments were set of each banana cultivar which contain: control (CK), inoculated with pathogen alone (TR4), banana leaves sprayed Routine(TR4+R2) Table 1.2.4The banana corms were dissected to investigate the disease index after 62 days post inoculated TR4. After plant corms were dissected, the degree of lesions of each plant corm was investigated according to five grades from 0 to 4. The five grades of 0, 1, 2, 3 and 4 represent no lesions of the corm, the area of corm lesions is 1-10%, the area of lesions in the corm is 11-30%, the area of corm lesions is 31-50% and the area of corm lesions is more than 50%, respectively. The formula for calculating the disease index and control effect is as follows .2.52>0.99).The roots and corms of bananas plants after inoculation with TR4 were frozen in liquid nitrogen immediately after being collected at 4 different time points, and then stored at -80°C for later use. Genomic DNA was extracted according to the cetyltrimethylammonium bromide (CTAB) method . Fungal 2.6For examined infection process and colonization of TR4, the roots and corms of the banana plants were collected after inoculated with TR4. Three biological replicates were designed for per treatment and 3 plantlets were prepared for per replicate. The samples were washed in sterile water and 75% alcohol, and cut into transverse and longitudinal thin slices with an ultra-thin blade. The slices were placed on the microscope slide with MQ water droplets, and then cover the sample with a glass cover slip. The processed samples were microscopically observed under a confocal laser scanning microscope . The spectral parameters of GFP fluorescence and plant autofluorescence in this confocal laser scanning microscope are  and , respectively.2.7Fresh corms of different treatments were collected at 1 d, 7 d, 14 d and 62 d after inoculation TR4. Three plants were prepared for each treatment as one biological replicate, and each treatment was repeated with three replicates. The corms were frozen and ground into powder in liquid nitrogen immediately after collection, and the starch content was determined using the Plant Starch Content assay Kit  according to the manufacturer’s instructions.2.8AGPase, GBSS, SBE) of banana in the starch synthetic pathway were selected for expression study at 1 d, 7 d, 14 d and 62 d post inoculation with TR4. Six defense-related gens NPR1, PR1, PR3, MYC2, ERF1 and ACC were also selected for this study. In each treatment, 9 corms were collected to detect the expression of related genes, and three technical replicates and three biological replicates were performed for each analysis. The collected corms sample were immediately frozen in liquid nitrogen and stored at -80°C, and then the total RNA was extracted using the Omega Plant RNA Extraction Kit according to the manufacturer’s protocols. The A260/A280 and A260/A230 of total RNA were 1.9 to 2.1 and 2.0 to 2.4, respectively, and can be used for further experiments. Additionally, cDNA was synthesized by the Prime Script RT Master Mix Kit (TaKaRa), and Reverse-transcription quantitative PCR (RT-qPCR) was performed using the iTaq Universal SYBR Green Supermix Kit (BIO-RAD) according to the manufacturer’s protocols. Relative changes in gene expression levels were calculated by the 2-△△CT method .Data were analyzed by using SPSS 25 and were graphed using Origin2018 (Graph Pad Software). All values are expressed as mean ± standard deviation, and statistically significant differences were determined using the Duncan multiple range tests , the hyphae were observed in the vascular bundle tissues only in cultivar ‘Baxi’ inoculated TR4 treatments, but not observed in other treatments. At 7 dpi to 62 dpi, a large number of hyphae were observed in the vascular bundle of root and it multiplies in large numbers in all treatment Figure 53.3.2To accurately understand the infection mechanism of pathogens, TR4 hyphae in the corms from 1dpi to 62 dpi were further monitored. Few TR4 hyphae were discovered in the cortex vascular tissues at 7 dpi and then expanded to the central cylinder in cultivar ‘Baxi’ inoculated TR4 treatments at 14 dpi. However, hardly any TR4 hyphae were observed in the cortex and central cylinder in other treatments. At 14 dpi, a mass of hyphae was found in the cortical root vessels of corms in the cultivar ‘Baxi’ inoculated TR4 treatments, while relatively few hyphae were found in the cortical roots of the other treatments. At 62 dpi, massive hyphae were observed in vessels of corms in cultivar ‘Baxi’ inoculated with TR4 only, while the level of pathogen hyphae in other treatments was relatively rare, so we turned our attention to the central cylinder of corms again. During this period, the central cylinder of the cultivar ‘Baxi’ inoculated TR4 treatments was ruptured, and many TR4 hyphae were released from the ducts to colonize the central column of the corms, while in other treatments, no hyphae released from the central column were found Figure 53.43.4.1TR4 hyphae were observed in both roots and corms of ‘Baxi’ (TR4 inoculated plants) one day after inoculation with TR4, while were not observed in ‘Yunjiao No.1’. From 1dpi to 7dpi, the number of hyphae in the root and corms of ‘Yunjiao No.1’ is lower than that of ‘Baxi’. After 14 dpi, quite a lot of hyphae were discovered in the roots of ‘Baxi’ and ‘Yunjiao No.1’. A mass of hyphae was also discovered in the corms in ‘Baxi’, and almost no hyphae were observed in ‘Yunjiao No.1’. These results indicated that the infection difference of TR4 in ‘Yunjiao No.1’ and ‘Baxi’ was mainly in the corms, and ‘Yunjiao No.1’ was more resistant than ‘Baxi’. Furthermore, quantity of hyphae in the root is higher than in the corms. These results show that the corms play an important role in blocking the infection of TR4 Figure 53.4.2One day after inoculated TR4, funguses were only discovered in the roots and corms of ‘Baxi’, but not in the tissues treated with isotianil. Numerous funguses were discovered in the roots and corms of all plants from 7dpi to 14dpi. However, a mass of funguses was discovered in the roots 62 dpi, while massively hyphae were observed only in the ‘Baxi’ without isotianil treatment in the corms, while ‘Yunjiao No.1’ and ‘Baxi’ treated with isotianil no mycelia were observed Figure 53.5NPR1, PR1, PR3, MYC2, ERF1 and ACC in corms was analyzed in the control (CK), TR4 inoculated plants (TR4) and isotianil treatment with TR4 inoculated plants . In both cultivars, isotianil application significantly induced or repressed the expression of PR1, PR3 and NPR1 in the pathways regulating salicylic acid at different time point. Compared with TR4 treatment alone, the expression of PR1 gene was significantly upregulated by routine application in ‘Baxi’ at different time point. The gene expression levels of TR4 inoculated plants (TR4) and isotianil application with TR4 inoculated plants  were as TR4-1 dpi: 4.70 ± 0.88; TR4-7 dpi: 0.36 ± 0.20; TR4-14 dpi: 7.83 ± 1.56; TR4+R1-1 dpi: 32.79 ± 7.50;TR4+R2-1 dpi: 38.87 ± 8.86; TR4+R1-7 dpi: 7.71 ± 2.46; TR4+R2-7 dpi: 5.14 ± 2.20;TR4+R-14 dpi: 19.47 ± 2.23; TR4+R2-14 dpi: 29.96 ± 9.44, respectively. In ‘Yunjiao No.1’, the PR1 gene expression levels of isotianil application with TR4 inoculated plants  were as TR4+R1-7 dpi: 8.83 ± 1.37; TR4+R2-7 dpi: 4.28 ± 0.81; TR4+R1-14 dpi: 3.27 ± 0.66; TR4+R1-62 dpi: 5.34 ± 1.41; TR4+R2-62 dpi: 3.14 ± 1.02, which were significantly upregulated than those of inoculation with TR4 alone , respectively. Additionally, compared with the TR4 treatment alone (TR4), the NPR1 gene expression of ‘Yunjiao No.1’ was significantly upregulated by isotianil application (TR4+R1 and TR4+R2), and the expression levels of were as TR4-7 dpi: 0.62 ± 0.09; TR4-14 dpi: 0.37± 0.09; TR4-62 dpi: 0.45 ± 0.14; TR4+R2-7 dpi: 1.22 ± 0.17; TR4+R1-14 dpi:1.28 ± 0.24; TR4+R2-14 dpi: 1.21 ± 0.45; TR4+R1-62 dpi: 1.11 ± 0.39; TR4+R2-62 dpi: 1.34 ± 0.54, respectively. In addition, the genes ERF1 that ISR-related genes were upregulated and downregulated as a result of the isotianil application in ‘Yunjiao No.1’ and ‘Baxi’, respectively. In ‘Yunjiao No.1’, the ERF1 gene expression levels of routine application were as TR4+R1-7 dpi: 9.68 ± 1.48; TR4+R2-7 dpi: 36.60 ± 6.65; TR4+R1-14 dpi: 4.45 ± 0.38; TR4+R2-14 dpi: 1.85 ± 0.29; TR4+R1-62 dpi: 76.05 ± 26.04, which were significantly upregulated than those of inoculation with TR4 alone , respectively. Additionally, the ERF1 gene expression of ‘Baxi’ were no significantly in TR4 treatment alone (TR4) and isotianil application treatment. Compared with the control treatment (CK), the ACC and MYC2 genes expression of ‘Yunjiao No.1’ were significantly upregulated by TR4 (TR4), and the expression levels of ACC and MYC2 were as TR4-7 dpi: 0.76 ± 0.09; TR4-14 dpi: 0.64± 0.13; TR4-62 dpi: 0.45 ± 0.08; TR4-1 dpi: 0.27 ± 0.03; TR4-7 dpi:0.45 ± 0.05; TR4-14 dpi: 0.73 ± 0.31; TR4-62 dpi: 0.25 ± 0.07, respectively, while there was no obvious trend in ‘Baxi’. All detailed results were presented in The relative expression of genes 3.6A microscopic observation analysis performed on root and corms samples from banana plants showed that tyloses plays an important role in against the pathogen infection in vascular bundle. At 62 days post inoculation (dpi), numerous tyloses were observed in the cortical root vascular bundle vessels of corms. At the same time, the hyphae were significantly reduced in the tissues with tyloses  related to starch synthesis in the corm were selected for its expression study, and the results showed that the expression of SBE, GBSS and AGPase in two cultivars was significantly different. These genes are significantly more expressed in ‘Yunjiao No.1’ than ‘Baxi’  when compared to TR4 inoculated plants. Isotianil pre-application significantly induced the expression of key genes PR1, PR3 and NPR1 of the SA pathway in banana plants. The result showed that isotianil may initiate the SA pathway to improve banana resistance to Fusarium wilt  and starch related  genes in ‘Yunjiao No.1’ are higher than those in ‘Baxi’. All the results demonstrated that ‘Yunjiao No.1’ is more resistant to FWB than ‘Baxi’, and resistant banana plants may prevent pathogenic fungal infection by inducing the content of tyloses in vascular bundles and starch grains in corms to form a physical barrier and activate immune pathways such as SAR and ISR in the corms.There are differences in the resistance of two cultivars. Our results showed that ‘Yunjiao No.1’ can significantly reduce the incidence of FWB and alleviate the disease symptoms much more than ‘Baxi’ cultivar. Comparing ‘Baxi’ with ‘Yunjiao No.1’, the disease index of ‘Yunjiao No.1’ was significantly lower than that of ‘Baxi’ Figure 3In the next step of our research, we will continue to explore the defense mechanisms of different resistant banana cultivars in the face of TR4 infestation, especially the relationship between tyloses, starch grain formation and plant resistance.5This study has shown that the plant elicitor isotianil can significantly reduce the impact of FWB and protect different banana cultivars. In addition, we further found that corms are important to against further infestation of TR4. The elicitor isotianil is able to induce the formation of tyloses in cortex vascular tissues of corms, preventing pathogen from root entering the corms. It can activate the three major systems of ISR, SAR, and starch granule synthesis in the corms, and inhibit the diffusion of pathogen in the corms, so as to reduce the effect of FWB. In addition, ‘Yunjiao No.1’ are more resistant than ‘Baxi’. In summary, when biological, chemical and agronomic measures are not ideal for the control of FWB, further enhance the resistance of cultivar together with elicitor isotianil application is a promising control strategy for banana growers.The original contributions presented in the study are included in the article/Conceptualization, G-DZ, SL and S-JZ. Methodology and software, G-DZ, PH and SL. Validation, G-DZ, PH, SL and S-JZ. Performed the experiment and data analysis, LT, SX, BY, LL, YW, TB and XL. Writing—original draft preparation, G-DZ, PH and SL. Writing—review and editing, SL and S-JZ. Supervision and project administration, SL and S-JZ. Project administration and funding acquisition, S-JZ. All authors have read and agreed to the published version of the manuscript.


As a method for sustainable agriculture, biofertilizers containing plant growth-promoting bacteria (PGPB) have been recommended as an alternative to chemical fertilizers. However, the short shelf-life of inoculants remains a limiting factor in the development of biofertilizer technology. The present study aimed to (i) evaluate the effectiveness of four different carriers  on the shelf-life of S2-4a1 and R2-3b1 isolates over 60 days after inoculation and (ii) evaluate isolated bacteria as growth-promoting agents for coffee seedlings.Coffea arabica). At 90 days following application, variables such as biomass and total N, P, K, Ca, and Mg uptakes of coffee seedlings were examined.The rhizosphere soil-isolated S2-4a1 and plant-tissue-isolated R2-3b1 were chosen based on their P and K-solubilizing capacities and their ability to produce IAA. To evaluate the alternative carriers, two selected isolates were inoculated with the four different carriers and incubated at 25 °C for 60 days. The bacterial survival, pH, and EC in each carrier were investigated. In addition, coconut coir dust inoculated with the selected isolates was applied to the soil in pots planted with coffee . The results of the present study suggested that coconut coir dust can be used as an alternative carrier for S2-4a1 and R2-3b1 isolates. The significant differences in pH and EC were observed by different carriers (P < 0.01) after inoculation with both bacterial isolates. However, pH and EC declined significantly only with coconut coir dust during the incubation period. In addition, coconut coir dust-based bioformulations of both S2-4a1 and R2-3b1 enhanced plant growth and nutrient uptake , providing evidence that isolated bacteria possess additional growth-promoting properties.The results showed that after 60 days of inoculation at 25 °C, the population of S2-4a1 and R2-3b1 in coconut coir dust carriers was 1.3 and 2.15 × 10 Coffee is one of the most significant industrial crops in Thailand, creating a revenue of more than 173.99 million USD annually. Arabica and Robusta are the most popular coffee varieties consumed worldwide. In Thailand, the bulk of Robusta coffee plantations are located in the south, accounting for 75% of the total Robusta coffee plantations, whereas Arabica coffee plants are located in the north . By focuSeveral investigations have demonstrated that the coffee rhizosphere is surrounded with a wide range of microorganisms . Some miMany of the PGPM have been isolated and have demonstrated their high performance under laboratory conditions. However, a lack of suitable carriers for biofertilizer, especially non-spore forming bacteria, influences the survival rates of inoculum, thereby reducing its efficiency after application into the soil. Carrier materials could provide suitable conditions for microbial colonization by building protected pore spaces and changing the soil physical characteristics . Most ofCocos nucifera L.), which remains after the coconut husk has been processed to produce the long fibers for industry, is the source of CCD. The mixture of CCD with other components has been widely and successfully used in many regions around the world as an environmentally friendly peat substitute for growing plants in the containers  has been taken into consideration as an alternative carrier utilized for the development of biofertilizers. The thick mesocarp, or husk, of the coconut fruit (ntainers . In addintainers . Consequntainers . TherefoCoffea arabica L.) were collected from abandoned coffee plantations (N18O 92′37N99O 32′78E), referring to coffee plantations without farmer management such as chemical fertilizer, pesticides, herbicides, and soil preparation. The average age of coffee plants was 17 years. Land use history was received by the interview from farmers.Rhizosphere soil and root tissue samples of coffee  3 g glucose, 2.5 g K2HPO4, 5 g NaCl, 3 g soy, 7 g trypton and 15 g agar with pH of 7.0 before their storage in a 40% glycerol solution at −80 °C until further analysis.Root samples were washed three times in sterile and distilled water, respectively. Subsequently, their surfaces were sterilized with 70% ethanol for 5 min and then in 2.6% sodium hypochlorite solution for 5 min. Finally, the samples were washed ten times with sterile distilled water to remove the NaOCl. The coffee roots were cut into one mm lengths and placed in a microcentrifuge tube contained 30 µL of sterile water. Root samples were softly ground by a stirring rod. Root suspension on the stirring rod tip was taken for streaking on tryptic soy agar (TSA) and incubated at room temperature for 7 days for bacterial colony formation. Colonies were selected and purified by subculturing three times on TSA media contained : 10 g glucose, 0.52 g K2HPO4, 0.41 g KH2PO4, 0.05 g Na2SO4, 0.2 g CaCl2, 0.1 g MgSO4 7H2O, 0.0025 g Na2MoO4 ⋅2H2O, 0.005 g FeSO47H2O and 15 g agar, and incubated at 30 °C for 7 days  contained (g Lr 7 days .3(PO4)2, for 72 h at 25 °C with constant shaking at 125 rpm. After 72 h, Pikovskaya (PVK) broth was centrifuged at 4,500 rpm for 10 mins, and supernatants were taken and checked for soluble phosphorus content in the culture broth using the molybdenum blue method  production. To determine the phosphorus solubilizing ability, the isolate was inoculated into 25 mL of pikovskaya broth (PVKB) contained: 5 g Cae method . As a coIAA production was determined by a colorimetric method . The bachttp://www.ncbi.nlm.nih.gov) using the Basic Local Alignment Search Tool (BLAST) software.S2-4a1 and R2-3b1 isolated from the coffee rhizosphere soil and root tissue, were chosen due to their ability to solubilize mineral phosphates and potassium. The genomic DNA of both bacterial isolates was extracted as described by 2 ≥75 wt%), vermiculite  and diatomite  were purchased from Shijiazhuang Huabang Mineral Products Co., Ltd. (China). While coconut coir dust (∼0.25 mm) was provided by the factory in Thailand. To prepare the carrier materials, 50 g of each carrier material was placed into a plastic bag, which was then sterilized by autoclave at 121 °C for 1 h. The carrier material was sterilized again 3 days after the first sterilization.The carrier materials used in this study were perlite, vermiculite, diatomite and coconut coir dust. Perlite  at 12 rpm at 28 °C for 7 days until reaching a concentration of 1 × 10Soil pH of all carriers were determined was measured by a pH meter using soil-water suspensions (1:5 v/v) and electrical conductivity (EC) by digital probe .Local Arabica coffee (Chiang Mai 80) seeds used in this study were surface-sterilized with 3% hydrogen peroxide for 3 min and subsequently rinsed with autoclaved distilled water five times. The sterilized seeds were sown into sterilized sand for 60 days after sowing (DAS), and then coffee seedlings of equal height were transplanted into black polyethylene bags (one plant per pot) filled with 1 kg of autoclaved media containing a mixture of soil and coconut coir dust based bioformulations (3:1 (w/w)) for the tests. The chemical analysis of the media for seedlings is presented in The coffee plants were destructively harvested after 90 days of treatment to determine the dry mass of leaves, branches and roots, which were then dried in an oven with forced-air circulation at 55 °C until a constant mass was obtained. From each plant tissue, 0.5 g of dry biomass was subjected to nitric-perchloric acid digestion to analyze total nitrogen (N), phosphorus (P), potassium (K), calcium(Ca) and magnesium (Mg) content . Total nIn this study, the nutrient transfer factor (NTF) was used as an indicator of the ability of the inoculated bacteria to improve nutrient uptake efficiency by transferring available nutrients into soil that can be directly absorbed by plants. The NTF was calculated by dividing the concentration of nutrients in the whole plants by the nutrient concentration in the media after the harvested plants. To determine the physico-chemical properties of the experimental media after harvest, the available phosphorus (P) of the media were determined by UV–VIS Spectrophotomete and the available K, Ca and Mg detection by ICP-OES .P value < 0.05 was considered for the significant level. Linear regressions were carried out to determine the spatial variations in the pH and EC and their changes with times. The interaction effects of carrier type and time on the bacterial population, pH, and EC were quantified using a two-way ANOVA. The statistical software used was SPSS 19.0 .Statistical analysis was conducted using analysis of variance (ANOVA). The least significant difference (LSD) was performed to compare the mean differences among treatments, and −1, respectively . While the highest population of R2-3b1 was found in the coconut coir carrier. At 45 and 60 DAI, there was no significant difference in the population of both S2-4a1 and R2-3b1 among carriers (P < 0.05), indicating that coconut coir dust could be used as an alternative carrier for developing the formulation of biofertilizer.A suitable carrier material should be able to stimulate and provide an environment for microbial development and the survival of the inoculated bacteria over long-term storage. In this study, the different carriers  inoculated with both bacterial S2-4a1 and R2-3b1 isolates were evaluated. The population of S2-4a1 inoculated in coconut coir dust, vermiculite and perlite and R2-3b1 inoculated in coconut coir dust, diatomite and vermiculite continuously increased during the first 30 days after inoculation (DAI) . The higP < 0.01)  in the present study  . The pH  < 0.01) . The pH  < 0.01) . The rannt study  and 3.P < 0.05), as shown in P < 0.01), pH (P < 0.05) and EC (P < 0.01). While the interaction of carrier and time had no significant effects on R2-3b1 population and pH. The impact of environmental factors on both S2-4a1 and R2-3b1 populations varied with carrier types . In addition, the inoculation of the S2-4a1 isolate increased the nutrient uptake of coffee seedlings  were tested for their growth enhancement effect on coffee seedlings. The results showed that inoculated treatments increased the total biomass of the seedlings compared to the uninoculated control . Howevereedlings , which iR2 = 0.59 P < 0.01), K (R2 = 0.95 P < 0.01), Ca (R2 = 0.75 P < 0.01) and Mg uptake (R2 = 0.89 P < 0.01). The plant biomass attained a positive correlation with the K (R2 = 0.85 P < 0.01), Ca (0.52 P < 0.01), and Mg  transfer factor but was not significantly correlated with the P transfer factor .Pearson’s correlation analysis was performed to determine the relationships between the plant P, K, Ca and Mg uptake, P, K, Ca, Mg transfer factor and plant biomass at 90 DAS . The plaP. chlororaphis strains 63-28 and TX-1 were higher when formulated in coconut fiber than in the other formulation materials . In addition, cocopeat had the ability to support the growth of two Pseudomonas sp. isolates  for up to 7 months with a population of 1 × 104 CFU g−1 as compared to talc and farmyard manure carriers  over time, which is a suitable alternative to diatomic, vermiculite, and perlite as carriers for bacterial inoculants. Consequently, employing coconut coir dust as PGPB carriers will enable the development of an efficient biofertilizer to meet Thailand’s biofertilizer demand. Furthermore, bioformulation prepared from coconut coir dust inoculated with the S2-4a1 and R2-3b1 isolates can affect and promote the nutrient uptake and growth of coffee seedlings, indicating the potential of these microorganisms for application as coffee inoculants and thus leading to a sustainable agriculture concept.10.7717/peerj.15530/supp-1Data S1Click here for additional data file.


Ka of 6.9, suggesting an involvement of the conserved histidine in proton transfer. The reaction rate depends on the nature of the divalent cation, likely bound by glutamate and asparagine side chains, and is log-linear with the metal ion pKa. We propose that the reaction is subject to general acid-base catalysis, using a combination of tyrosine and histidine acting as general base and water directly coordinated to the metal ion as general acid. The reaction is temperature dependent; activation energy Ea = 37 kcal mol−1, suggesting that cleavage is coupled to opening of DNA in the transition state.ANKLE1 is a nuclease that provides a final opportunity to process unresolved junctions in DNA that would otherwise create chromosomal linkages blocking cell division. It is a GIY-YIG nuclease. We have expressed an active domain of human ANKLE1 containing the GIY-YIG nuclease domain in bacteria, that is monomeric in solution and when bound to a DNA Y-junction, and unilaterally cleaves a cruciform junction. Using an AlphaFold model of the enzyme we identify the key active residues, and show that mutation of each leads to impairment of activity. There are two components in the catalytic mechanism. Cleavage rate is pH dependent, corresponding to a p  Caenorhabditis elegans resulting from exposure to ionizing irradiation . A deleted form beginning at methionine 331 (ANKLE1331–615) was found to be fully active, and used in all the experiments described in this work. This was found to contain a mutation R492H that facilitated growth in E. coli. Site directed mutation of active site residues were performed by PCR using the same Q5 site directed mutagenesis kit and the appropriate primers , cloned into pGEX6P, using the Q5 Site-Directed mutagenesis kit (New England Biolab E0554S). DNA primers were chosen to delete sequences between the N-terminus and chosen positions . PCR pro primers . All DNAE. coli were transformed with pGEX6P-ANKLE1331–615 R492H and its mutants and grown in LB supplemented with 100 mg/l carbenicillin and 20 mg/l gentamicin at 37°C until the OD600 of the culture reached 0.6. The temperature of the culture was lowered to 12°C. When the absorbance of the culture at 600 nm was between 1.0 and 1.5, ANKLE1331–615 R492H production was induced by the addition of IPTG to a final concentration of 200 μM and incubated for 24 h. Bacterial cells were harvested by centrifugation at 7500 g for 15 min at 4°C. The cell pellet was resuspended in 40 ml/l of cell culture of 25 mM HEPES (pH 7.5), 1M NaCl, 5% glycerol, 0.5 mg/ml lysozyme and 1 tablet of complete protease inhibitors (Roche) and incubated at room temperature for 15 min. Triton X100 was added to a final concentration of 0.2% (w/v) and the extract was then sonicated on ice at 30 W for 1.5 min using 10 s pulses separated by 30 s pauses using a FB705 sonicator (Fischer Scientific). The cellular extract was clarified by centrifugation at 20 000 g for 30 min at 4°C (Beckman rotor JA25.50). The supernatant was mixed with glutathione sepharose 4B beads , which were preequilibrated with 25 mM HEPES (pH 7.5), 1 M NaCl, 5% glycerol. The beads were then washed with the same buffer. GST-ANKLE1331–615 R492H was eluted as a fusion protein by incubating the beads with 25 mM HEPES (pH 7.5), 50 mM NaCl, 5% glycerol and 20 mM glutathione. Alternatively, GST-ANKLE1331–615 R492H was cleaved with human 3C protease while bound to the glutathione column overnight in 25 mM HEPES (pH 7.5), 150 mM NaCl, 5% glycerol. GST fusion or non-fusion ANKLE1331–615 R492H were further purified by chromatography on Fractogel SO−3 (Merck) in 25 mM HEPES (pH 7.5), 5% glycerol with a linear NaCl gradient from 50 mM to 1 M, followed by purification on Mono S 5/50 GL  in 25 mM HEPES (pH 7.5), 10% glycerol, 0.5 mM EDTA with a linear NaCl gradient from 50 mM to 1 M.Arctic Express −1 cm−1 for ANKLE1331–615 R492H or 79 600 M−1 cm−1 for GST-ANKLE1331–615 R492H and stored at –70°C.Protein concentration was measured by UV spectrophotometry using an extinction coefficient of 36 500 M331–615 R492H purity was analysed by polyacrylamide gel electrophoresis in denaturing condition. Protein samples were subjected to electrophoresis in 10% polyacrylamide Bis–Tris NuPAGE (Invitrogen) in 50 mM MOPS, 50 mM Tris (pH 7.7), 3.47 mM SDS, 1 mM EDTA. Protein bands were detected using Instant Coomasie Protein Stain (Abcam) and electrophoretic migration was compared to standard proteins .ANKLE1Oligonucleotides were purchased from Sigma-Aldrich.32P]-labelled by polynucleotide kinase (Thermo Scientific). 2 μM of oligonucleotide was incubated with 0.17 μM [γ-32P]-ATP at 37°C in 50 mM Tris (pH 7.6), 10mM MgCl2, 5 mM DTT, 0.1mM spermidine for 30 min. 50 mM EDTA was added to terminate the kinase reaction before the addition of the other oligonucleotide in order to generate the splayed Y-junction substrate. Oligonucleotides were hybridized to generate splayed Y-junctions by slow cooling from 80°C to 4°C. The substrate was purified by electrophoresis in 8% (29:1) polyacrylamide gels in 90 mM Tris–boric acid (pH 8.3), 2 mM EDTA.Splayed Y-junction was prepared by the hybridization of r- and x-strands . Where r331–615 R492H was subjected to gel filtration on Superdex 75 (10/300) at a flow rate of 0.4 ml min−1 in 25 mM HEPES (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 10% glycerol. Elution of ANKLE1331–615 R492H was compared to that of carbonic anhydrase  with an estimated molecular mass of 29 000 DaANKLE12, 0.1 mg/ml BSA. ANKLE1331–615 R492H Y453F was added to the indicated concentrations. Fluorescence anisotropy (r) was measured as :The r-strand was synthesized with fluorescein attached at the 3′ terminus, using a 6-fluorescein CPG column (Glen Research 20-2961). The splayed Y-junction was generated by hybridization with the h-strand of J3. Fluorescent anisotropy measurements were performed with a SLM Aminco 8100 fluorescence spectrometer. The excitation wavelength was selected using an interference filter of 482.5 ± 31 (fwhm) nm, and the emission was selected using a monochrometer set at 530 nm with slits set at 8 (fwhm) nm. Fluorescence was measured at 20°C using 100 nM splayed Y-junction in 20 mM HEPES–NaOH (pH 7.0 or 8.0), 50 mM KCl, 2 mM MnClIVV and IVH are fluorescence intensity with the polarizers parallel and crossed respectively, and G is the ratio of detector sensitivity between vertical and horizontal polarization, and was measured for each determination in these studies.where The data were fitted to the following binding model :r0 is the anisotropy in the absence of protein, Δr is the change in anisotropy over the full range of protein concentration, Kd is the dissociation constant for ANKLE1 binding to the splayed Y-junction, [Ank] and [Y] are the ANKLE1331–615 R492H and splayed Y-junction DNA concentrations respectively.where AlphaFold (model Q8NAG6). The overall confidence of the model for the nuclease domain (residue 448–566) of Ankle1 was mostly very high (64%) and the positioning of the backbone of the residues of the mutants in this study were either very high  or confident (H498 and N565).The structure of Ankle1 was predicted by the software 32P] x-strand labelled splayed Y-junction DNA was preincubated with 2 μM ANKLE1331–615 R492H (or active site mutant) in 20 mM cacodylate (pH 6.5), 50 mM KCl, 0.1 mg/ml BSA at 37°C. After 3 min incubation, MnCl2  was added to a final concentration of 10 mM to initiate the cleavage reaction. Aliquots were removed at chosen times and the reaction terminated by addition of an equal volume of 25 mM EDTA, 95% formamide. DNA substrate and products were separated by electrophoresis in a 20% (19:1) polyacrylamide gel electrophoresed in 90 mM Tris–boric acid (pH 8.3), 2 mM EDTA, 8 M urea. Radioactivity was detected by exposure to a storage phosphor screen, and quantified using a BAS-1500 phosphorimager (Fuji), or a Typhoon FLA 9500 fluorimager . Reaction progress was fitted by non-linear regression analysis to single exponential functions using KaleidaGraph (Abelbeck Software).5 nM radioactively--labelled on the indicated strand was pre-incubated at room temperature for 5 min in the presence of 0, 20 nM, 200 nM or 2 μM ANKLE1331–615 R492H in 10 mM cacodylate (pH 6.5), 50 mM KCl, 50 μg/ml calf thymus DNA and either 1 mM EDTA (for the active enzyme) or 10 mM MgCl2 (for the Y453F mutant). The 20 μl reactions were initiated by adding 2 μl of a 12.5 mM KMnO4 solution and terminated by addition of 1.5 μl of β-mercaptoethanol after 1 min. Reaction products were precipitated by addition of 1/10 volume of 3 M sodium acetate and 3 volumes of ethanol, resuspended in 100 μl of 1 M piperidine and incubated at 95°C for 30 min. Samples were dehydrated by addition of n-butanol and removal of the solvent phase, followed by vacuum desiccation. Dried samples were resuspended in formamide and analysed on denaturing 15% polyacrylamide sequencing gels. These were dried, exposed to storage phosphor screens and visualised with a Typhoon FLA 9500 fluorimager . Purine sequence markers were generated by subjecting the same substrates to reaction with formic acid (100 nM splayed Y-junction DNA radioactively--labelled on either the x-strand (the 5′ terminus of the duplex) or the r-strand (the 5′ terminus of the single-stranded flap) and incubated with no protein, or with 20 nM, 200 nM or 2 μM ANKLE1331–615 R492H in the presence of 1 mM EDTA, or ANKLE1331–615 R492H Y453F in the presence of 10 mM MgCl2 . This is fully consistent with the demonstration that the enzyme cleaves the four-way junction of a supercoil-stabilized cruciform structure unilaterally. Thus although a four-way junction is a substrate for ANKLE1, the result is a junction containing a single cleaved strand rather than complete resolution. Given that the N-terminal region of hANKLE1 that is not present in the 331–615 fragment has ankyrin repeats this could mediate protein-protein interactions that might be important in its function. However, the observation that hANKLE1331–615R492H acts in monomeric form is consistent with the primary substrate being the splayed Y-junction or similar structure, and ANKLE1 is most active on that branchpoint. Many GIY-YIG nucleases act in monomeric form  and central (Y453) strands. These are flanked above and below by helices on which are displayed the key histidine H498, glutamate E551 and asparagine N565. This arrangement of protein structure and the disposition of critical side chains is typical of a number of GIY-YIG nucleases that have been determined structurally . The putLike the majority of junction-selective nucleases, ANKLE1 catalyses the hydrolysis of a specific phosphodiester linkage in a DNA junction, with transfer of the phosphate to the 5′-terminus that is created Figure . In prinKa = 6.9. That would be consistent with a histidine undergoing proton transfer in the transition state, and the pH profile of the H498A mutant is substantially altered. In the structure predicted by AlphaFold, N2 of H498 is 2.9 Å from the phenolic oxygen of Y453. A very similar arrangement of tyrosine and histidine was observed in the crystal structures of Eco29kI  Human endonuclease ANKLE1 localizes at the midbody and processes chromatin bridges to prevent DNA damage and cGAS-STING activation. Adv. Sci. 10, e2204388.gkad416_Supplemental_FileClick here for additional data file.