In whole-cell patch clamp on cell line- and bone marrow-derived osteoblasts, ZOL potentiated outward currents. On RAW264.7, ZOL (10−4 M)-evoked current was reduced by the Kv channel blocker tetraethylammonium hydrochloride (TEA), but not by the selective TRPV1-channel antagonist capsazepine. On MC3T3-E1 cells and bone marrow-derived osteoblasts, ZOL-evoked current (5 × 10−8 to 10−4 M) was reduced by capsazepine, whereas the selective TRPV1-channel agonist capsaicin potentiated the control current. In the cell proliferation assay, 72 h incubation of RAW264.7 and MC3T3-E1 cells with ZOL reduced proliferation, with IC50 values of 2.62 × 10−7 M and 2.02 × 10−5 M, respectively. Mineralization of MC3T3-E1 cells and bone marrow-derived osteoblasts was observed in the presence of capsaicin and ZOL (5 × 10−8–10−7 M); ZOL effects were antagonized by capsazepine. In summary, the ZOL-induced activation of TRPV1 channel mediates the mineralization of osteoblasts and counterbalances the antiproliferative effects, increasing the IC50. This mechanism is not operative in osteoclasts lacking the TRPV1 channel.Bisphosphonates (BPs) reduce bone pain and fractures by balancing the osteoblast/osteoclast ratio. The behavior of ion channels in the presence of BPs is not known. To investigate this, the effect of zoledronic acid BP (ZOL) (3 × 10 Bone tissue is one of the preferred sites for metastasis in patients with advanced malignancy, especially in case of prostate, breast cancer, and multiple myeloma. Tumor cells prefer to migrate into heavily vascularized areas of the skeleton, such as the red marrow of the long bones, sternum, pelvis, ribs, and vertebrae , becauseIt is noteworthy that once cancer has spread to bone tissue, it can rarely be cured. The current standard of care for patients with bone loss due to osteolytic bone metastases includes antiresorptive therapy that can shrink or slow the growth of bone metastases and contribute to reducing bone pain and the risk of pathologic fractures, but that is not curative . Among tBesides the beneficial effects deriving from the interaction of the BPs with osteoclasts, these drugs also show antimyeloma and antitumor activity, leading to the increased overall survival of patients with various malignancies . Indeed,−9 M to 10−6 M [−5 M) [2+ levels in osteoblasts, as well as their maturation and differentiation [Although the effects of BPs for therapeutic intervention in bone metastases appear to be linked to the modulation of osteoclast activity and reduction of tumor cell proliferation, in the last years there has been increasing interest in BPs’ effects on osteoblasts and osteocytes, which led to uncovering their capability to promote increased mineralization, thereby improving bone density and thickness. In fact, BPs can prolong osteoblast lifespan , inducinM [−5 M) . Some exM [−5 M) . More rentiation . Despite+ channels and transient receptor potential (TRP) channels.It is now evident that a wide variety of factors are involved in the balance between bone resorption and bone production. In full agreement with this, a large variety of ion channels are known to play a leading role in regulating bone homeostasis, including K+ (Kir) channels, and small-, intermediate-, and large-conductance Ca2+-activated K+ channels [2+-activated K+ channels contribute to the regulation of cell volume, resting membrane potential, and intracellular levels of Ca2+ ions, essential for the mineralization process [Ca3.1 calcium-dependent potassium channels cause membrane hyperpolarization required to optimize membrane potential during Ca2+ entry, maintaining the driving force for Ca2+ entry that activates stem cell proliferation [+ channel Kir2.1 impairs both osteoblastic and chondrogenic processes, leading to the rare Andersen’s syndrome [2+-activated K+ (BK) channels are involved in cell proliferation, since the incubation of SH-SY5Y cells with BK channel antagonists paxilline, iberiotoxin, and resveratrol was associated with a reduction of cell proliferation, reducing cell diameter and inducing AKT1pser473 dephosphorylation [Mesenchymal stem cells, the precursors of osteoblasts, are known to express different potassium channels which include KATP channels, inward rectifier Kchannels . Ca2+-ac process . Moreove process . For exaferation . Insteadsyndrome . Furtherrylation . 2+ and Mg2+. Some specific TRP channels, including TRPM7, TRPV1, TRPV5, and TRPV6, appear to be involved in cellular Ca2+ and Mg2+ ion influx, thus regulating proliferation, differentiation, secretion, and apoptosis processes [2+/Mg2+ ions homeostasis [Osteoblasts were also shown to express different kind of TRP channels, a very large family of cation-non-selective ion channels, differentially permeable to Carocesses . TRPV1, rocesses . Furtherrocesses ; TRPV1 arocesses . It was rocesses . More rerocesses . Furtherrocesses , therebyrocesses . TRPM7 geostasis . −8 to 5 × 10−4 M) on ion channels, especially potassium and TRP ion channels, cell proliferation, and mineralization were investigated on RAW264.7 preosteoclast-like cells, MC3T3-E1 preosteoblast-like cells, and rat/mouse native bone marrow-derived osteoblasts, and the obtained results are described below.Despite these considerations, BP effects on ion channels and the possible correlation between current modulation and BPs’ therapeutic effects on bone cell survival and osteoblast activity—essential for preserving bone homeostasis also in the presence of metastases, maintaining bone density and thickness and reducing bone pain sensation—have never been reported. The effects of zoledronic acid (ZOL) (3 × 10−8 to 5 × 10−4 M), to evaluate their capability in inducing antiproliferative effects on osteoclasts and pro-survival effects on osteoblasts.We first compared the effects of three clinically used bisphosphonates , risedronate (RIS), and zoledronic acid (ZOL)) on preosteoclast-like cells RAW264.7 and preosteoblast like cells MC3T3-E1 at different concentrations (3 × 10F = 1.123). The maximal efficacy against RAW264.7 was, however, in favor of ZOL vs. the other BPs, with ZOL being more effective in inhibiting cell proliferation than ALE, as evaluated by Student t-test comparing the Emax percentage (%) of the different drugs (p < 0.05) (F = 1.111). The Hill coefficient was <1 for all the compounds in RAW264.7, whereas a slope >1 was calculated for MC3T3-E1. In MC3T3-E1 cells, all BPs caused a mild but not significant increase of dehydrogenase activity in the nanomolar concentration range (3 × 10−8 to 10−7 M)  . Also, i 10−7 M) a,b.−4 M, the compounds were all capable of reducing the numbers of differentiated osteoclasts . In our previous work, these concentrations were effective in inducing mineralization in cell lines [−8 M, ZOL was the most effective drug in inducing mineralization, which increased by +136.08% ± 21.48% compared to controls (number of replicates = 5) as determined by Student t-test (p < 0.05). At this concentration, RIS and ALE were less effective than ZOL in inducing nodule formation, causing an increase of +65.63% ± 5.22% and +58.78% ± 6.08% vs. controls group (p < 0.05) (number of replicates = 3), respectively. Nodule formation of calcium phosphate precipitate was visible after 10–15 days of incubation of cells with drugs in the mineralized medium  of these cells was −44.49 ± 3.40 mV (number of cells = 48). The application of iberiotoxin (Ibtx), a selective BK channel blocker, to a final concentration of 4 × 10−7 M, reduced the cationic currents inducing cell depolarization. The co-incubation of the Ibtx with 5 × 10−3 M TEA did not induce a further reduction of the cationic currents, suggesting that BK channels carried at least 27.9% of the control currents. The co-incubation of the cells with Ibtx, TEA, and 5 × 10−3 M Ba2+ caused an almost complete reduction of the control currents, suggesting the contribution of inward-rectifying potassium (Kir) channels and outward-rectifying K+ channels to the control currents . No effect of ZOL was observed on the current amplitude at nanomolar concentrations.Under this experimental condition, 10voltages d. In detIn agreement with our functional observation, the mRNA levels and protein of TRPV1 gene was not detected by others in this cell line ,29,30.−3 M reduced the current amplitude, and co-incubation with 5 × 10−3 M Ba2+ caused an incomplete reduction of voltage-dependent currents, suggesting the presence of other channels, beside BK and Kv channels, contributing to the control current  channel agonist, elicited large outward currents. Ruthenium red (RR), a non-selective TRP channel blocker, completely reduced the capsaicin-induced currents at 10−5 M, supporting the idea that the TRPV1 channel is functionally active in these cells . At −80 mV (Vm) the current amplitude was −18.2 ± 0.49 pA (number of cells = 45), while a large outward current of +105.1 ± 24.9 pA (number of cells = 45) was recorded at positive membrane potentials. TEA at 5 × 10 current a. The apse cells b.−5 M and 10−4 M ZOL increase outward currents. ZOL-evoked currents were almost completely abolished after the application of 10−5 M ruthenium red  , and the inward current at −80 mV (Vm) by +12.1%.The application of ZOL on the macroscopic currents of MC3T3-E1 cells induced a concentration-dependent potentiation of these currents. Both 10nium red c. ZOL at mV (Vm) c. ZOL at− solution, 10−4 M ZOL applied to the cells confirmed its ability to potentiate outward currents, which could be partially reduced by 10−5 M ruthenium red by −55.39%, and were fully reduced after the application of TEA (5 × 10−3 M) and Ba2+ (5 × 10−3 M) (−4 M ZOL was still capable of inducing a notable activation at membrane voltages >+80 mV (Vm). In particular, 10−4 M ZOL enhanced currents by +4.46% at +30 mV (Vm), significantly enhanced current amplitude by + 54.83% at +60 mV (p < 0.05) and +725.08% at +100 mV (Vm) vs. controls (p < 0.05) (In low Cl 10−3 M) f. Under  < 0.05) h.+-channel blockers Ba2+ (5 × 10−3 M) and TEA (5 × 10−3 M) in the Cl−-free bath solution. We found that the application of ZOL (10−4 M), even in the presence of these blockers, potentiated the outward currents that were antagonized by ruthenium red (10−5 M), indicating that ZOL-activated currents were carried not only by K+ channels, but also by other different cationic channels, possibly TRP  caused an enhancement of the outward currents (number of cells = 10). In the majority of the investigated cells, the ZOL-evoked current was markedly reduced by TEA (5 × 10−3 M) by −66.36% at +100 mV (Vm); a further and full reduction of the current was observed with capsazepine (10−6 M) (−7 M) were also responsive to capsaicin (10−5 M). Instead, no activation was observed in cells not responsive to capsaicin (data not shown) after a long-term incubation time with ZOL (10−7 M). These findings indicate that currents activated by ZOL (10−7 M) involved TRPV1 and/or Kv/BK channels.Since ZOL, at 10(10−6 M) a. Cells −8 M) on the same MC3T3-E1 cells. After about 10 min of incubation time, ZOL (5 × 10−8 M) caused an enhancement of the outward currents (number of cells = 16). In the majority of the investigated cells, the ZOL-evoked current was markedly reduced by TEA (5 × 10−3 M) by −60.91% at +100 mV (Vm), with a further reduction of the current by −15.42% at +100 mV (Vm) after the application of capsazepine (10−6 M) (−8 M ZOL failed to activate the current. These findings indicate that 5 × 10−8 M concentration could be the threshold ZOL concentration to potentiate the Kv/BK and TRPV1 channels in the MC3T3-E1 osteoblast cell line in whole-cell configuration. Similar effects were observed after the application of ZOL (5 × 10(10−6 M) b. In somIn agreement with this observation, the mRNA level of the TRPV1 gene was found to be elevated in this cell line and osteoblasts ,32.+ concentration in the bath and pipette solutions, a hyperbolic current was observed that crossed the voltage axis −40 ± 8.28 mV (number of cells = 15) and −36.08 ± 3.78 mV (Vm) (number of cells = 12), representing the resting potential of the mouse and rat cells, respectively. TEA (5 × 10−3 M) as well as capsazepine (10−6 M) reduced current amplitudes to control values suggesting the contribution of Kv/BK and TRPV1 channels to the total current in either cell population (−5 M) potentiates the hyperbolic current, supporting the idea that TRPV1 channels are functionally active in these cells  isolated from both mouse and rat bone marrow . Firstlypulation a–d. The se cells b–e. Both−4 M) elicited large, outward currents that were blocked by co-application of capsazepine (10−6 M) and TEA (5 × 10−3 M), further confirming ZOL’s capability of enhance TRPV1 and Kv/BK channel currents (−4 M) enhanced current amplitude by +5.37% at −30 mV, +29.82% at +30 mV, and +47.69 at +50 mV vs. controls, and on rat MSCs by +6.94 at −30 mV, +101.37% at +30 mV, and +212.67% at +50 mV. On rat, TEA (5 × 10−3 M) caused a reduction of −75.35%, and capsazepine (10−6 M) completely closed ZOL-evoked currents, supporting the idea that Kv/BK channels are responsible for most of the ZOL-activated currents in these cells (−4 M) also enhanced the inward current by +81.9% at −90 mV in mice and +54.3% in rat, and these effects were fully inhibited by TEA and capsazepine  with or without channel inhibitors.The effects of ion channels modulators on the mineralization process induced by ZOL was evaluated using the mineralization assay both in mouse and rat bone marrow-derived osteoblasts a,b and M−8 to 5 × 10−3 M) and capsazepine (5 × 10−8 to 10−7 M) on ZOL-induced mineralization were investigated at different concentrations after 15 days of incubation of the culture in the mineralized medium. The co-incubation of TEA (5 × 10−8 M) with ZOL (5 × 10−8 to 10−7 M) reduced the capability of ZOL to induce mineralization in 4 out of 10 experiments on rat and mouse bone marrow-derived osteoblasts. TEA reduced the ZOL-induced mineralization by −29.64% ± 8.52% (number of experiments = 4). Also, the co-incubation of capsazepine (5 × 10−8 M) with ZOL (5 × 10−8 to 10−7 M) reduced the capability of ZOL to induce mineralization in 8 out of 10 experiments on these cells. Capsazepine antagonized the ZOL-induced mineralization by −29.50% ± 5.01% (number of experiments = 8).On native bone marrow cells, the effects of TEA (5 × 10−6 M) alone on murine bone marrow cells was responsible for inducing strong mineralization leading to an increase of +209.19% ± 45.33% (number of experiments = 3) compared to the control conditions, suggesting a key role for the TRPV1 channel in mineralization and the osteoblastogenesis process. NaF (10−3 M) a well-known chemical inducing mineralization agent that led to a mineralization increase of +215% ± 58.39% (number of experiments = 3) compared to the control condition on mouse bone marrow-derived osteoblast cells , can be explained by the fact that the threshold concentration of ZOL for inducing TRPV1-mediated mineralization is 10−7 M. This is also the threshold concentration required to activate TRPV1 and Kv/BK channels in osteoblasts in our experiments. Maximal mineralization is observed at 10−7 M of ZOL and, under these conditions, capsazepine is fully effective. Moreover, capsaicin (10st cells a. The fa−3 M) on mouse bone marrow-derived osteoblast cells. We found that ZOL at 5 × 10−8 and 10−7 M concentrations was less effective than NaF (10−3 M) in inducing mineralization. In these experiments, capsazepine was more effective than TEA in antagonizing the ZOL-induced mineralization on mouse bone marrow-derived osteoblast cells  reduced the proliferation at high concentrations while, at 5 × 10−8 M, this compound did not significantly affect proliferation. It is evident that TEA alone at high concentrations caused a reduction of cell proliferation, thereby affecting mineralization after 15 days of incubation time. TEA at 5 × 10Xenopus oocytes; it was found that ZOL was capable of activating TRPV1 currents when directly applied to the excised patch (−8 M) enhanced TRPV1 currents of +51.25% at +30 mV, +21.58% at +60 mV, and +32.06% at +100 mV (Vm) with respect to the current activated by capsaicin (10−5 M) at the same voltages. Therefore, ZOL at nanomolar concentration activates the TRPV1 channel current expressed in Xenopus oocytes, in excised macropatches, at negative and positive membrane voltages.Finally, the effects of ZOL on TRPV1 channels were evaluated by using excised inside-out macropatches in ed patch . In partIn the present work, we show that zoledronic acid, at nanomolar concentrations, potentiates outward currents and induces cell proliferation and mineralization of MC3T3-E1 preosteoblast-like cells, and rat and mouse native bone marrow-derived osteoblasts. This drug at micromolar concentrations also has antiproliferative effects on RAW264.7 preosteoclast-like cells and MC3T3-E1 preosteoblast-like cells.+ channels. These findings were supported by the fact that ZOL is capable of potentiating the whole-cell currents recorded in MC3T3-E1 cell and the mouse and rat bone marrow-derived osteoblasts, and these currents were partially antagonized by the selective TRPV1 blocker capsazepine and by the unselective Kv/BK channel blocker TEA. The ZOL activating action of the outward currents was observed in physiological conditions, as well as in Cl−-free bath solution, supporting the notion that the currents were carried by cations. Furthermore, the zoledronic acid-induced mineralization was prevented by capsazepine in 8 out of 10 experiments by −29.5%. TEA was also effective in 4 out of 10 experiments in antagonizing the mineralization induced by ZOL by −29.4%. These findings suggest that Kv/BK and TRPV1 channels contribute to the ZOL-induced mineralization by about 60%, while the residual 40% of mineralization is sustained by mechanisms involving, for instance, the activation of connexin 43 and the inhibition of intracellular protein tyrosine phosphatases [The zoledronic acid-induced mineralization, observed at nanomolar concentrations, is mediated by the activation of TRPV1 channel with the contribution of TEA-sensitive voltage-dependent Kphatases ,13. Also, it was not possible to use specific BK channel blockers, such as Ibtx or paxilline, because of their irreversible blocking action of the BK channel associated with antiproliferative effects ,34. Also−8 M) on TRPV1 channel appears to be mediated by the direct interaction with the channel subunits, as demonstrated by the fact that this drug was capable of activating, in excised macropatches, the TRPV1 channel expressed in Xenopus oocytes. Currently, we do not know the location of the binding sites of ZOL on the channel subunits modulating channel function. It can be speculated that the binding site is located on the cytosolic face of the TRPV1 channel. This hypothesis is supported by the fact that ZOL, at nanomolar concentrations, is more effective in activating the TRPV1 current of a clone expressed in Xenopus oocyte in excised macropatches, rather than the TRPV1 whole-cell current recorded in the osteoblasts. Also, the activating effect of ZOL on the TRPV1 channel current at nanomolar concentrations is rescued following the long-term incubation of the osteoblasts with the drug.The action of ZOL , the BPs failed to increase intracellular dehydrogenase activity on RAW264.7 cells in the cell proliferation assay. These findings suggest that the observed ZOL-activating action of TRPV1 channel is associated with cell proliferation and mineralization of osteoblasts with cytoprotection, explaining its selective action as an antiproliferative drug against the osteoclasts lacking TRPV1 channel current. Moreover, in the nanomolar concentration range  and provided by Sigma-Aldrich . Cells were provided at P4 and used from P5 to P10. Differentiated MC3T3-E1 cells for the mineralization assay were cultured in the mineralization medium (MM) obtained by the addition of 50 µg/mL ascorbic acid and 10 mM β-glycerophosphate  [Preosteoblast-like cells from mouse MC3T3-E1 (ECACC 99072810) and preosteoclast-like cells from mouse RAW264.7 (ECACC 91062702) were cultured in Minimum Essential Medium Eagle-Alpha Modification (αMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% antibiotics (penicillin–streptomycin), under standard conditions, at 37 °C, in a humidified atmosphere containing 5% COMO, USA) ,37.2. Non-adherent cells were carefully removed after 3 h of incubation time. After primary cultures had become almost confluent, they were treated using Trypsin-EDTA Solution 1×  for 2 min at room temperature. The same protocol was applied 3 times, so that a purified population of MSCs was obtained 3 weeks after the initiation of culture, and then used for experiments. Mouse and rat bone marrow-derived osteoblasts for the mineralization assay were cultured in the mineralization medium.Native mouse and rat mesenchymal stem cells were obtained as previously described . BrieflyXenopus laevis oocytes were performed at Centro Interdisciplinario de Neurociencia de Valparaíso (CINV) (Chile) in accordance with NIH “Guidelines for Egg and Oocyte Harvesting in Xenopus laevis” [In accordance to 3R RULE (DIRECTIVE 2010/63/EU) Replace, Reduce, Refine, bone marrow cells were collected both from wild-type male mice C57BL sacrificed for other scientific purposes  and from male Wistar rats . Patch clamp experiments on  laevis” .3 cells/well, and pre-incubated for 24 h under standard conditions. Then, cells were treated for 72 h with different concentrations of the drug solution. Finally, the activity of the intracellular dehydrogenases was evaluated by adding CCK-8 solution (10 μL) to each well and measuring the absorbance at λ = 450 nm after 2 h of incubation with CCK-8 solution, using the microplate reader Victor® 3V . The changes of the cell viability were expressed as percentage (%) changes of intracellular dehydrogenase activity induced by the drug with respect to the control.The activity of the intracellular dehydrogenases was evaluated by using the Cell Counting Kit-8 (CCK- 8) (Sigma-Aldrich), a highly sensitive test that utilizes water-soluble tetrazolium salt ,39. WST-6 cells/mL/well) were cultured in the mineralization medium using 12- and 24-well plates in the presence of different concentrations of the drug solution for approximately 10–15 days under standard conditions. The medium was refreshed every 3 days. After mineralized nodules have become visible, cells were stained with the alizarin red S staining solution 2% (m/v), able to form a bright red stained calcium complex, and washed 3 times with PBS to avoid non-specific dying. For the quantification, 10% acetic acid solution was added to each well to completely elute the dye, and then the absorbance at λ = 405 nm was measured using the microplate reader Victor® 3V. The changes of mineralization ability were expressed as the percentage (%) change of mineralization induced by drugs with respect to the control or with respect to 10−3 M NaF, a well-known chemical compound inducing mineralization [This assay was performed to evaluate cells’ capability to mineralize, that is, their ability to create calcium deposits or nodules after the expression of a mature osteoblast phenotype, as previously described . Brieflylization . For co-4 cells/well) were incubated in 6-well plates containing glass slides to allow photos to be taken at the end of the experiment in a specific medium of differentiation containing 50 ng/mL RANKL. The medium was refreshed every 2 days. After 7 days of differentiation with RANKL, cells were treated with different concentrations of the drug solutions for 72 h; then, they were fixed with 10% formaldehyde/PBS for 15 min, and the plate was washed using deionized water and then stained for TRAP, according to the manufacturer’s instructions. The staining of randomly selected areas was evaluated at the microscopy and thereby photographed. The TRAP kit  was used to evaluate the ability of drugs to inhibit osteoclast proliferation and activity by detecting the presence of tartrate-resistant acid phosphatase in mature osteoclasts. RAW264.7 cells -N,N,N′,N′-tetraaceticacid (EGTA), 10 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 1 mM Na2ATP, 0.3 mM Na2GDP (pH = 7.2). The bath solution contained 142 mM NaCl, 2.8 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 11 mM glucose, and 10 mM HEPES (pH = 7.4) [−, 142 × 10−3 M of NaCl was replaced by Na-glutamate. CaCl2 was added to the pipette solutions to give a free Ca2+ ion concentration of 1.6 × 10−6 M in whole-cell experiments. The calculation of the free Ca2+ ion concentration in the pipette was performed using the MaxChelator software  [Whole-cell patch clamp experiments on RAW264.7 cells, MC3T3-E1 cells, and native mesenchymal stem cells (MSCs) from bone marrow of mouse and rat were performed in asymmetrical KH = 7.4) ,41. For CA, USA) .Xenopus oocytes expressing TRPV1 channel, the same solution was used both for the pipette and external recording solution: 150 mM NaCl, 10 mM EGTA, 2 mM MgCl2, and 10 mM HEPES (pH = 7.4) [For macropatch experiments on H = 7.4) .2+ ions, ruthenium red (RR), capsaicin (Caps), and capsazepine (Capsz) were all purchased from Sigma . The three bisphosphonates (BPs), zoledronic acid (ZOL), alendronate (ALE), and risedronate (RIS), were synthetized and purified in our labs as previously described [Channels modulators iberiotoxin (Ibtx), tetraethylammonium hydrochloride (TEA), Baescribed .2+, and Capsz were prepared by dissolving the drugs in dimethyl sulfoxide (DMSO), RR was solubilized in water, and Caps was dissolved in ethanol, at concentrations of 118.6 × 10−3 M. BPs were provided as a stock solution in phosphate-buffered saline (PBS) at 1 or 10 mM. Stock solutions were stored in the refrigerator, whereas dilute solutions were directly prepared on the day of the experiments and maintained at room temperature. For cell proliferation assays, diluted solutions of drugs were prepared using the medium. For patch clamp experiments, microliter amounts of the stock solutions were added to the bath solutions as needed. DMSO did not exceed 0.07%; at this concentration, this solvent does not normally affect current or cell proliferation. Stock solutions of Ibtx, TEA, Ba+ ion concentrations  using whole-cell patch clamp technique. For mesenchymal stem cells from mouse and rat bone marrow cells, the current was obtained in response to voltage pulses from −90 to +70 mV (Vm) in 20 mV steps. Currents are expressed as densities (pA/pF) to control for cell size/capacitance differences. All experiments were performed at room temperature (20–22 °C) and sampled at 2 kHz (filter = 1 kHz) using an Axopatch-1D amplifier equipped with a CV-4 head-stage . Patch pipettes were pulled from PG52165-4 Patch Clamp #8250 Glass Capillaries, 1.65mm o.d., 1.1 mm i.d. (World Precision Instruments) with a vertical puller  to give a resistance of 8–10 MΩ. Data acquisition and analysis was performed using pCLAMP 10 software suite  as previously described [Drug actions on the channel currents recorded during instantaneous I/V relationships were investigated by applying a depolarization protocol, in a range of potentials going from −100 to +100 mV (Vm) for RAW264.7 cells, and from −80 to +120 mV (Vm) for MC3T3-E1 cells in 10 mV steps, starting from HP = −60 mV (Vm), in physiological conditions, with asymmetrical Kescribed ,46,47. SXenopus oocytes, the transcribed cRNA for TRPV1 channel was injected into oocytes using a Nanoliter 2010 WPI  microinjector. Then, they were incubated in ND96  solution at 18 °C for 2–3 d after injection [For patch clamp experiments in njection . Patch pnjection ,49.Axopatch 200B system (Axon Instruments) using Clampex 10 (Axon Instruments) acquisition software. Leak subtraction was performed based on a P/4 protocol [Inside-out excised patch recordings were performed at 20° using patch pipettes with a tip size of 15–25 µm (pipette resistance <1 MΩ after polishing). Currents were obtained in response to voltage pulses from −60 to +260 mV (Vm) in 10 mV steps and were recorded with an protocol . Electrot-test was used to evaluate the significance of differences between the means of two groups. p values < 0.05 were considered to indicate statistical significance.Data were collected and analyzed using Excel software (Microsoft Office 2010); curve fitting was made using SigmaPlot 10.0. The statistical results are presented as mean ± SEM. The number of replicates relative to each experimental dataset was reported in the results paragraph and in the figure description. The Student Xenopus oocytes, the percentage of activation of ZOL was calculated as (I drug − I CTRL)/(I Caps − I CTRL), referring to the amount of currents activated by the TRPV1-agonist capsaicin (10−5 M) at the same voltages.For patch clamp experiments on cells, the percentage of activation was calculated as (I drug − I CTRL)/(I max − I CTRL), where I max was evaluated at the maximum voltage applied in the control (CTRL) condition. For patch clamp experiments on TRPV1 channel can be a novel target for zoledronic acid and structurally related BP compounds, mediating the proliferative cytoprotective action of these drugs on osteoblasts. Further investigations are needed to clarify the binding site/s of this class of drugs on the TRPV1 channel. This mechanism may contribute to the anti-osteoporotic effect of zoledronic acid, as well as the pain-relieving effect. Beneficial effects of zoledronic acid can be expected in skeletal muscle where TRPV1 channel appear to play a role in activating hypertrophic signaling and cytoprotection ,52.Currently no patents are resulting from the work reported in this manuscript.


The 2010 Infectious Diseases Society of America (IDSA) guidelines for management of cryptococcal diseases recommend high dose fluconazole (≥ 800 mg/day), either alone or with other antifungal drugs, as alternative anticryptococcal choices. But evidence for its use in the treatment of HIV-uninfected cryptococcal meningitis (CM) remains sparse.A retrospective analysis of HIV-uninfected CM patients who received fluconazole 800 mg/day for salvage therapy from January 2011 to December 2016 at Huashan Hospital, Shanghai, China was performed. Efficacy and safety were assessed, and mortality and prognostic factors evaluated.A total of 44 patients were studied including 19 refractory to amphotericin B induction therapy, 8 refractory to fluconazole consolidation therapy (400 mg/d), and 17 intolerant of antifungal drugs. For salvage, 11 patients received triple therapy of high dose fluconazole, amphotericin B and flucytosine, 20 received dual therapy of high dose fluconazole and flucytosine, 13 received monotherapy of high dose fluconazole. Median duration of high dose fluconazole in salvage regimens was 136.5 days . Clinical response rates were 72.1% (31/43) and 83.7% (36/43) when assessed at 2 weeks and the end of salvage therapy, respectively. Adverse events possibly related to high dose fluconazole occurred in 54.5% (24/44) of the patients, and all were mild or moderate. From the initiation of salvage therapy, 1-year all-cause mortality was 13.6% (6 of 44 patients) among the study population with no significant difference in refractory or intolerant patients.Adherence to guideline recommendations of high dose fluconazole, alone or in combination with other antifungals, was safe and often effective for salvage therapy of HIV-uninfected CM patients. Cryptococcus neoformans, while Cryptococcus gattii is more geographically restricted [Cryptococcal meningitis (CM) is the most common opportunistic fungal infection of the central nervous system (CNS). The vast majority of cases are caused by stricted . Accordistricted . Owing tstricted . Howeverstricted . Mortalistricted –8. As a Cryptococcus. It has been widely used in CM as consolidation and maintenance therapy, usually at a dosage equal to or lower than 400 mg/day. In resource-limited areas, fluconazole alone has been used for induction therapy, and proved to be a poorly effective drug with 10-week mortality consistently > 50% even at high dosage [Fluconazole is a triazole with excellent in vitro and in vivo activity against h dosage –12. But h dosage –18. In th dosage , 20. HowThis is a retrospective cohort study conducted among HIV-uninfected CM patients from January 2011 to December 2016 at Huashan Hospital, Fudan University . Patients who had been refractory to or intolerant of prior antifungal drugs and who then switched to salvage therapy with fluconazole 800 mg/day were included. This study was reviewed and approved by the local medical ethics committee.Cryptococcus from cerebrospinal fluid (CSF), (2) positive CSF ink smear, (3) cryptococcal capsular polysaccharide antigen detected in CSF on cryptococcal antigen (CrAg) lateral flow assay  in CSF or (4) compatible histopathological findings, which are 5–10 μm encapsulated yeasts in brain tissue. Refractory CM was considered if any 2 of the following conditions were present after an adequate period of antifungal therapy (≥14 days): (1) persistently positive cultures of Cryptococcus in CSF, (2) deterioration of clinical signs and symptoms of disease, (3) new sites of disease or worsening of pre-existing lesions radiologically, and (4) decreasing level of glucose and increasing level of protein in CSF [A proven diagnosis of CM was made if the patient met any of the following criteria: (1) positive culture of n in CSF , 22. PatPatients treated with high dose fluconazole for ≥1 week were included for efficacy evaluation. Response to salvage therapy was assessed at 2 weeks after initiation of salvage therapy, at the end of salvage therapy, and at the end of antifungal therapy. Efficacy of antifungal treatment was categorized as success  or failure  on the basis of clinical, radiological, and microbiological data according to previous criteria . AdverseP-value of < 0 .05 was considered statistically significant.Continuous variables were compared with t test or the non-parametric Mann-Whitney test. Proportions were compared with the χ2 test or Fisher’s exact test, as appropriate. Log-rank test was used in univariate analysis for prognostic factors of 1-year survival, and Cox proportional hazards model for identification of independent prognostic predictors. A In total, 44 patients with proven CM were included in our study. Thirty-two also had pulmonary involvement and 6 cryptococcemia. The median age was 44 years, and 63.6% were male. Predisposing factors co-existed in 16 patients (36%). Detailed information is summarized in Table Cranial magnetic resonance imaging (MRI) was performed in all patients, with 41/44 (93.2%) yielding abnormal results. Local parenchymal lesions (86.4%) were most frequently observed, and common sites included frontal lobe (68.2%), parietal lobe (59.1%), periventricular region (29.5%) and basal ganglion (22.7%). Other abnormalities included meningeal enhancement  and ventricular enlargement . Most patients had 2 or more sites of involvement. Abnormalities on chest computer tomography (CT) scans were found in 32 of 44 patients (72.3%). The common findings were nodules and masses (47.7%), patches and strips (43.9%), pleural thickening (22.7%), pleural effusion (20.5%), lymph node enlargement (6.8%), air bronchogram sign (4.5%) and ground glass attenuation (2.3%). Some patients had mixed types of lesion on chest CT.Cryptococcus in 4 patients, and ink smears were positive in 18 patients. CSF CrAg tests were positive in all 43 patients, and 47.7% of them (21/43) had CrAg titer of at least 1:640. Median white blood cell count (WBC) of CSF was 29 /mm3 . Median levels of CSF protein and glucose were 824 mg/dL and 34 mg/dL, respectively. Of the 42 patients who had lumbar punctures, 21 had CSF opening pressure above 25 cm H2O.Before initiating salvage therapy, CSF examinations were performed in 43 patients including 42 samples from lumbar punctures, 1 from continuous lumbar cerebrospinal fluid drainage. CSF cultures were positive for As listed in Fig. Of the 19 patients refractory to initial therapy, 11 received high dose fluconazole in combination with AmB and flucytosine for salvage, 6 received high dose fluconazole and flucytosine, and the remaining 2 received high dose fluconazole alone. For patients who were refractory to consolidation therapy, fluconazole dosage was increased from 400 mg/day to 800 mg/day and flucytosine co-administered in all but one patient. Of the 17 antifungal-intolerant patients, 10 received high dose fluconazole alone, and 7 others were treated with high dose fluconazole and flucytosine. Overall, triple therapy of high dose fluconazole, AmB and flucytosine was administered in 11 patients, 20 received dual therapy of high dose fluconazole and flucytosine, 13 received monotherapy of high dose fluconazole. Median duration of high dose fluconazole in salvage therapy for the 44 patients was 136.5 days . Among the 31 patients (70.5%) receiving flucytosine, 27 were treated with flucytosine throughout the course, and the remaining 4 discontinued the drug for suspected side effects . Median duration of flucytosine in salvage therapy for the 31 patients was 142 days .P = 0.019), there was no apparent differences in efficacy at the end of salvage therapy  and the end of antifungal therapy . At 1-year follow-up after stopping antifungal drugs, all of the 37 successfully treated patients survived, and no relapses were observed.Except 1 patient who received high dose fluconazole less than 7 days and died of multiple organ failure (MOF), the other 43 patients were all included into efficacy evaluation. Two weeks after salvage therapy, partial response in 31 patients 72.1%), stable response in 11 patients (25.6%), and death caused by brain hernia in 1 patient (2.3%) were observed. At the end of salvage therapy, 3 patients (7.0%) achieved complete response, 33 (76.7%) achieved partial response, 2 (4.7%) achieved stable response, and 5 (11.6%) died. The overall efficacy rate was 83.7%. Of the 2 stable patients, 1 discontinued fluconazole, reinstituted with AmB and flucytosine and achieved partial response; the other was lost for follow-up 40 days after initiation of salvage therapy. Of the 4 patients who died after 2 weeks of salvage therapy, 1 was considered to have died of CM, 2 died from secondary infection and MOF, and 1 died from aggravation of lymphoma. Thirty-seven of 43 patients (86.0%) achieved clinical success and stopped antifungal drugs, 5 (11.6%) died and 1 (2.3%) was lost for follow-up. As shown in Table .1%, stabAll 44 patients were included in the safety assessment. Possible high-dose fluconazole related AEs were found in 54.5% (24/44) of them. No grade 3 or 4 AEs were observed. As shown in Table In terms of clinical manifestations, gastrointestinal side effects were common complaints. Skin lesions were documented in 2 patients. Photosensitive dermatitis occurred in 1 patient soon after initiation of fluconazole, and improved without fluconazole discontinuance. Rash developed in another patient several weeks after the salvage therapy and lasted until CM recovered and antifungal treatment finished. We also observed 1 patient with elevated D-dimer and deep venous thrombosis which was relieved after treatment with oral warfarin. None of these patients discontinued high dose fluconazole due to intolerance.P = 0.258). We assessed factors with a potential impact on all-cause mortality by means of univariate and multivariate analyses. In the univariate model, factors significantly associated with 1-year mortality were age ≥ 60 years (P = 0.001); serum albumin level < 35 g/L (P = 0.001); estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73m2 (P = 0.015); and CSF WBC count < 10 /mm3 (P = 0.002). CSF CrAg titer ≥1280 was more frequently presented in mortality group but it did not reach a statistic significance (P = 0.058). By multivariate analysis, as shown in Table P = 0.014) and CSF CrAg titer ≥1280 (P = 0.048).From the beginning of salvage therapy, 1-year mortality related to all cause was 13.6% (6 of 44 patients) with no significant difference among 3 treatment groups (Fig. In 1996, Pappas et al. reported 12 HIV-uninfected CM patients who received high dose fluconazole, 800 mg/day, in their induction therapy or as consolidation therapy . HoweverAmB-based regimens are recommended to optimize fungal clearance and survival for both HIV-infected and HIV-uninfected CM patients . DespiteIn our study, no significant difference in 1-year mortality rates was found among patients with refractory infections and those intolerant of prior antifungal drugs. Factors linked with poor prognosis in our patients were older age, renal dysfunction, lower CSF white cell count, and high CSF CrAg titers, as consistent with previous reports in HIV-uninfected population , 25, 33.In previous studies of treatment for cryptococcal or other fungal infections, fluconazole was well tolerated at dosages from 800 to 2000 mg/day , 34–37. There were some limitations to this work. As we included a limited number of cases in the present study and did not include a group of patients who received low dose fluconazole as a control, the results should be interpreted with caution. Only a few patients were culture positive when changed to salvage therapy. For these patients, the reasons for “failure” may be due to the poor efficacy of previous therapy, dysfunctional immune responses, or associated underlying conditions, and some may have done well if the previous antifungal therapy had been continued. Hence, multicenter randomized controlled trials are needed for making a further objective evaluation.Evidence for the use of high dose fluconazole in HIV-uninfected CM patients remains sparse. Our study specifically addressed the efficacy and safety of high dose fluconazole in this population and suggested that fluconazole 800 mg/day alone, or in combination with other antifungal drugs was a safe and promising choice for HIV-uninfected CM patients. Either refractory cases or those intolerant to prior antifungal drugs can achieve favorable outcomes after receiving high dose fluconazole as salvage therapy.


Catalpa fargesii Bur is a valuable commercial tree in China and identifying SNPs that associate with wood property would make a foundation of the marker-assisted breeding in the future. However, related work has not been reported yet.Association study is a powerful means for identifying molecular markers, such as single-nucleotide polymorphisms (SNPs) associated with important traits in forest trees. SUS) gene from the genome of C. fargesii, which is a key enzyme in sucrose metabolism and also associated to wood formation in trees, coding 806 amino acids that expressed mainly in young branches, xylem, and leaves according to real-time quantitative PCR. Then we identified allelic variations of CfSUS associated with nine wood quality associated traits in Catalpa fargesii Bur. Totally, 135 SNPs were identified through cloning and sequencing the CfSUS locus from a mapping population  and 47 of which were genotyped as common SNPs  in the association population that comprised of 125 unrelated individuals collected from main distribution area. Nucleotide diversity and linkage disequilibrium (LD) analysis showed CfSUS has a relative low SNP diversity (πT = 0.0034) and low LD (r2 dropped below 0.1 within 1600 bp). Using the association analysis, we found 11 common SNPs and 14 haplotypes were significantly associated with the traits , explaining 3.21–12.41% of the phenotypic variance. These results provide molecular markers above associated with wood basic density, pore rate, and six other traits of wood, which have potential applications in breeding of Catalpa fargesii Bur.We cloned a 2887 bp long sucrose synthase  contains supplementary material, which is available to authorized users. Catalpa fargesii Bur. is a valuable tree native to China. Its timber not only exhibits good mechanical properties, such as stiffness and ultimate stress, but also has good chemical properties and high corrosion resistance [C. fargesii breeding. Although the mechanisms influencing wood properties are unclear, some researchers have speculated that the specific structure, content, arrangement, and interaction of macromolecules in secondary cell walls may confer unique properties, making wood better suited for different applications [sistance . The proications . This suications , 3.PtoPsbW gene and five wood quality traits have been identified in Populus tomentosa [Populus deltoides significantly influenced eight composite traits that associated with wood properties, including lignin percentage, lignin syringyl/guaiacyl ratio, lignin structure, and the growth of plants [Molecular marker-assisted selection has been proven effective in resolving the complex quantitative traits of genetic components to improve and accelerate traditional tree breeding. In particular, association mapping, is an effective way for elucidating the potential relationship between allelic variation and complex quantitative trait variations in natural populations . Previouomentosa . In addif plants .SUS participates in providing UDP-glucose for cellulose biosynthesis and directly associated with cellulose synthase complexes [SUS may help identify molecular markers associated with wood quality traits to guide C. fargesii breeding.Sucrose is the main source of carbon for compounding cellulose, one of the major components of secondary cell walls, and wood vascular tissue in the stem comprises highly active sink cells that use sucrose for cellulose synthesis . In thesomplexes . TherefoC. fargesii (CfSUS). Real-time quantitative PCR (RT-qPCR) was used to identify the gene expression in six different organs. Subsequently, nucleotide diversity and linkage disequilibrium decay within the CfSUS were assessed in a mapping population (n = 93). Finally, single-marker- and haplotype-based association tests were performed to examine the putative effects of allelic variations on nine wood quality traits in an association population comprising 125 C. fargesii individuals. This is the first time that association analysis about CfSUS were studied to identify molecular markers probably affecting wood property, which could support the genetic improvement of C. fargesii and other tree species.In this study, we first cloned and identified a SUS gene family member in C. fargesii individuals that collected from all of the provenances throughout its whole natural distribution range in China, for the initial SNP association mapping. The distribution zone from which these individuals were collected, could be divided into four geographic regions: Fenhe River Valley, Jinghe River Valley, Jialingjiang River Valley, and Yellow River Valley. In the year 2009, branch segments of 125 native C. fargesii individuals were collected from eight cities in four provinces and grafted, and the population was grown in Xiaolongshan National Nature Reserve, Gansu Province, China . The clonal plantation was established using a randomised complete block design with two plants per clone in each block (row spacing is 2 m and plant spacing is 2 m) and totally six replicates. Within the association population, 93 individuals were randomly selected  to identify SNPs within the gene via PCR amplification and sequencing.We used a population consisting of 125 unrelated The 125 individuals of the association population were sampled in 2012 to characterise their wood quality traits. Cores from bark to pith were collected in the south-facing direction of the original stems to measure wood density and other wood properties with an increment borer (7 mm) at breast height (1.3 m above the ground). The wood samples were fixed in formalin–acetic acid–alcohol (FAA) after collection, and nine wood property traits were measured in 2013: wood basic density (WBD), pore rate, cell wall percentage , cell wall thickness, radial lumen diameter, chordwise lumen diameter, radial fibre central cavity diameter, chordwise fibre central cavity diameter, and average fibre central cavity diameter.2/(W1 − W2 + W2/ρcw), where W1, W2 and ρcw represent the water-saturated weight, oven dry weight, and wood cell wall component density, respectively. We used the constant 1.53 g/cm3 for ρcw [WBD was measured according to the formula WBD = W for ρcw , 12. The for ρcw : Cores f for ρcw . The phe for ρcw .The meanCfSUS, including a 2418-bp open reading frame (ORF) from a previous RNA sequencing data. We amplified CfSUS cDNA using SUS-CDS-specific primers  according to the manufacturer’s instructions. First-strand cDNA was synthesised from 2 g RNA using PrimeScript™ 1st Strand cDNA Synthesis Kit . We obtained the 2887-bp complete coding sequence(CDS) of CfSUS based on the cDNA sequence on the conserved domain . Finally, 5067 bp genomic DNA sequences of CfSUS, including a 1394-bp 5′UTR, 3406-bp coding region, and 267-bp 3′UTR were obtained. The entire DNA sequence of CfSUS was verified using the SUS-e primers . Four specific primers  were designed to sequence the introns in CfSUS was used for BLAST in the GenBank database and multiple SUS proteins from other species were selected for phylogenetic tree construction using MEGA software and alignment with DNAMAN software. We analysed the phylogenetic relationship of CfSUS with the amino acid sequences of SUS from other species identified from NCBI (http://www.ncbi.nlm.nih.gov) using BLAST : Orobanche ramosa (AEN79500.1), Nicotiana tabacum (AHL84158.1), Solanum tuberosum (NP_001274911.1 and NP_001275286.1), Solanum lycopersicum (CAA09681.1 and ADM47608.1), Cichorium intybus (ABD61653.1), Actinidia chinensis (AFO84090.1), Camellia sinensis (AHL29281.1), Gossypium hirsutum (ADY68848.1), Gossypium barbadense (ADY68844.1), Arachis hypogaea var. vulgaris (AEF56625.1), Jatropha curcas (AGH29112.1), Gossypium aridum (AEN71079.1), Gossypium tomentosum (AEN71067.1), Eucalyptus grandis (ABB53602.1), Populus tomentosa (ADW80558.1), Manihot esculenta (ABD96570.1), Hevea brasiliensis , Hordeum rulgare (CAA46701.1 and CAA49551.1), Lolium perenne (BAE79815.1), Triticum aestivum (CAA04543.1 and CAA03935.1), Bambusa oldhamii , Oryza sativa , Saccharum officinarum (AAF85966.1), Zea mays (AAA33514.1), and Tulipa gesneriana (CAA65639.1). Phylogenetic tree was conducted using MEGA ver. 5(maximum likelihood method) [Amino acid sequences of cum AHL848.1, SolaCfSUS in different tissues using RT-qPCR.Total RNA was extracted from six different tissues, including leaf, bark, phloem, xylem, flower, and young branch, from three 11-year-old “Xianhuiqiu” clone (as three times repetition) using RNeasy Kits  and reverse-transcribed into cDNA using PrimeScript™ 1st Strand cDNA Synthesis Kit . The cDNA samples were used to analysis the expression of −ΔΔCT method.RT-qPCR was performed with a LightCycler 480 System  using the SYBR Premix Ex Taq Kit  with the recommended amplification system by the manual. The primers for amplification , DNA sequences including 53-bp 3′UTR and 3406-bp coding region, was cloned, sequenced, and analysed in 93 randomly selected individuals from the C. fargesii association population. To ensure the sequencing accuracy, four pairs of primers  were used to amplify four fragments (800–1500 bp) of the whole sequence   and DnaSCfSUS was estimated by linear regression analysis of LD using DnaSP v4.90.1. The LD level between the 47 common SNP markers were valued as r2  using the HAPLOVIEW (https://www.broadinstitute.org/haploview/haploview), where the interval of the parameter varies from 0 to 1. The significance of r2  for all the SNP sites were calculated using 100,000 permutations. Genotypic data of CfSUS identified in this population were showed in Additional file The decay of LD with the increase of physical distance between SNPs within the candidate region of n = 125), 47 common SNPs and nine wood properties associated traits were considered, and a mixed linear model (MLM) was selected to fit each SNP-phenotype combination using TASSEL v2.0.1 [Qυ + Zu + e. In this formula, “y”, “μ”, “υ”, “u” and “e” respectively represent a vector of phenotype observation, (a vector of) intercepts, (a vector of) population effects, (a vector of) random polygene background effects and (a vector of) random experimental errors. Q is a matrix used to define the population structure by STRUCTURE and Z is a matrix relating y to u. Var(u) = G = σa2κ with σa2 is the unknown additive genetic variance and κ is the kinship matrix. In this model, the Q and K represent the estimated membership probability and pairwise kinship, respectively. The Q matrix was identified based on the population structure pattern (K = 3) within the association population , which was assessed by STRUCTURE v2.3.1 [R2) explained by each SNP was calculated use the formula R2 = SSt/SST, where SSt and SST represented the variance between genotypes and the total variance, respectively [The associated mapping was carried on according to the method of Wang et al. . In the L v2.0.1 . We usedE v2.3.1 . The K mE v2.3.1  and usedHaplotype frequencies and haplotype association tests were evaluated and performed using Haplotype Trend Regression software  on a three-marker sliding window. The significance of the associations was tested using 1000 permutation. Only haplotypes with a frequency not less than 1% were selected and positive FDR (Q ≤ 0.1) was used to correct the multiple test.d) to additive(a) effects(|d/a|) estimated from the least-square means for each genotypic class. The algorithm and formulas used for calculating dominance (d) and additive(a) effects were described by Du et al. [d/a| ≤ 0.5 was defined as additive effects, whereas partial or complete dominance was defined as the values within the range 0.50 < |d/a| < 1.25, and values of |d/a| ≥ 1.25 were regarded as overdominance.The modes of gene action were defined as the ratio of dominant , a 202-bp 5′UTR sequence, and a 267-bp 3′UTR sequence. The full length of the CfSUS DNA sequence was 5067 bp, containing a 3406-bp coding region flanked by a 1394-bp 5′UTR sequence and 267-bp 3′UTR sequence  ranged from 0 (exons 2 and 12) to 0.0103 (intron 3), while θw ranged from 0 (exons 2 and 12) to 0.0241 (intron 3) in coding regions.To characterise the nucleotide diversity and linkage disequilibrium of 2 value dropped to 0.1 in less than 1600 bp .The degree of LD showed a linear regression at a relatively rapid rate; the r bp Fig. , indicatP < 0.05 and Q < 0.10) . In this case, the value of |d/a| was 0.189 and appeared to be additive effect (Table 2 = 6.39%) and the mode of gene action was appeared to dominant effects  using MLM to identify the single-SNP-based associations. In total, 17 significant associations with eight phenotypic traits  were identified , were significantly associated with seven traits  , the nucleotide diversity (πT = 0.0034) of CfSUS was similar to that of CfC3h , suggesting that these two genes share a similar pattern of genetic variance in natural C. fargesii populations. However, the nucleotide diversity of CfSUS was lower than that of PtSUS1 (πT = 0.0092) and PtSUS2 (πT = 0.0109), which had been proved as potential homologous gene in present study in Populus tomentosa [SUS gene may be more conserved in C. fargesii than P. tomentosa.SNP-based association analyses are important for elucidating the SNP distribution and frequency patterns within a candidate gene . In thisCfSUS was rapidly decayed within 1600 bp , which indicated that low LD may also existed in other genes of C. fargesii. However, few studies have assessed the LD in C. fargesii, and the degree of LD in other genes or the whole genome of C. fargesii is unknown. In future studies, we will estimate LD decay in more genes and longer genomic fragments and even explore the haplotype variability on the whole-genome level.Understanding LD patterns is indispensable for association mapping. In our study, LD in omentosa , Eucalyps nitens , Pinus slvestris , loblolllvestris , and Doulvestris , which slvestris . MoreoveCfSUS gene , which indicated that CfSUS have a potential utility of a gene-based association mapping method in developing SNP markers in C. fargesii. Finally, we identified 11 SNPs and 14 haplotypes were significantly associated with wood property by association analysis. These findings imply a functional role of CfSUS in mediating wood properties and providing SNP markers associated with wood property, suggesting the potential application in marker-assisted breeding of C. fargesii in the future.In this study, we cloned a Additional file 1Table S1. Phenotype of associated population. (XLSX 19 kb)Additional file 2Figure S1. The distribution pattern for each trait measured in the entire population (n = 125) of Catalpa fargesii. (JPG 7743 kb)Additional file 3Table S2. The mean values, range of variation (RV), standard error (SE) and coefficient of phenotypic variation (CV (%)) for each wood property trait measured in association population. (DOCX 18 kb)Additional file 4Table S3. Primers used in this paper. (DOCX 15 kb)Additional file 5Table S4. Genotypic data of CfSUS identified in the associated population. (XLSX 30 kb)Additional file 6Figure S2. Genomic organization of CfSUS. Positions of 47 common SNP markers are shown as vertical lines. (JPG 1318 kb)Additional file 7Figure S3. Phenotypes of different genotypes of significant SNPs associated with wood property traits. (JPG 6446 kb)Additional file 8Figure S4. An unrooted phylogenetic tree of SUS members from Arabidopsis thaliana, Populus trichocarpa and Catalpa fargesii. Arabidopsis thaliana: ATSUS1(AT5G20830.1), ATSUS2(AT5G49190), ATSUS3(AT4G02280), ATSUS4(AT3G43190), ATSUS5(AT5G37180) and ATSUS6(AT1G73370); Populus trichocarpa: PtrSUS1(Potri.018G063500.1), PtrSUS2(Potri.006G136700.1), PtrSUS3(Potri.002G202300.1), PtrSUS4(Potri.015G029100.1), PtrSUS5(Potri.012G037200.1), PtrSUS6(Potri.004G081300.1), PtrSUS7(Potri.017G139100.1); Catalpa fargesii Bur.:CfSUS. (JPG 1588 kb)


The role of complexation in biological fractionations of silicon isotopes is more complicated, likely involving several enzymatic processes and active transport proteins. The integrated understanding greatly strengthens the potential of δ30Si proxy for reconstructing the paleo terrestrial and oceanic environments, and exploring the meteorites and planetary core formation, as well as constraining ore deposits and hydrothermal fluid activity.The fundamental advances in silicon isotope geochemistry have been systematically demonstrated in this work. Firstly, the continuous modifications in analytical approaches and the silicon isotope variations in major reservoirs and geological processes have been briefly introduced. Secondly, the silicon isotope fractionation linked to silicon complexation/coordination and thermodynamic conditions have been extensively stressed, including silicate minerals with variable structures and chemical compositions, silica precipitation and diagenesis, chemical weathering of crustal surface silicate rocks, biological uptake, global oceanic Si cycle, etc. Finally, the relevant geological implications for meteorites and planetary core formation, ore deposits formation, hydrothermal fluids activities, and silicon cycling in hydrosphere have been summarized. Compared to the thermodynamic isotope fractionation of silicon associated with high-temperature processes, that in low-temperature geological processes is much more significant . The equilibrium silicon isotope fractionation during the mantle-core differentiation resulted in the observed heavy isotope composition of the bulk silicate Earth (BSE). The equilibrium fractionation of silicon isotopes among silicate minerals are sensitive to the Si–O bond length, Si coordination numbers (CN), the polymerization degrees of silicate unites, and the electronegativity of cations in minerals. The preferential enrichment of different speciation of dissoluble Si (DSi) (e.g., silicic acid H After td Taylor , and thod Taylor . In the d Taylor ,45,46,47d Taylor , archivid Taylor ,17,18), d Taylor ,50,51,52d Taylor ,55,56,5730Si data, multiple bio-geochemical proxies, and parameters) and potential challenges  for silicon isotope geochemistry studies [During the initial study period from the 1950s to 1990s, the progresses of silicon isotope geochemical studies have been well demonstrated by Ding et al. . The rec studies . In this4+ [In early studies, the silicon isotope compositions were measured by using the gas-source mass spectrometry (GS-MS), whereby the silicon was analyzed in the form of the volatile SiF4+ . Then, t4+ ,64,65. M4+ .4. The first one is to digest the solid rocks/minerals in hydrofluoric acid (HF), followed by precipitation with BaCl2 as BaSiF6. The precipitate is thermally decomposed into gaseous SiF4 to transfer to the GS-MS [4 by adding the mixture gas of F2 and HF as the fluorinating agent [4 by using the hazardous chemicals of BrF5 or F2 [4-GS-MS approach is as high as ±0.1‰ (2σ) [4 by using the acid decomposition of Cs2SiF6, providing an analytical precision generally better than ±0.15‰ (2σ) [4-GS-MS approach is greatly limited because of obvious disadvantages, such as complicated sample preparation procedure, time-consuming nature, and use of extremely hazardous and fluorine-based chemicals [For the analysis of silicon isotope compositions using the gas-source mass spectrometry (GS-MS), there are four methods for transforming solid silicates into gaseous SiFhe GS-MS . With thng agent . The thiF5 or F2 , which h.1‰ (2σ) . Fourthl15‰ (2σ) . Howeverhemicals ,44.The in situ analysis of silicon isotopes has been achievable with the secondary ion mass spectroscopy (SIMS) since the 1980s ,64,65,6914N16O+), and the recoveries >98% indicate that silicon losses through polymerization are insignificant. This technique is able to determine relative Si isotope variations of samples with a long-term external reproducibility of ±0.14‰ (2σ) [42−) and dissolved organic carbon (DOC), which induce shifts in the silicon isotope composition. An improved procedure is to remove SO42− by forming barite precipitation and to remove DOC by the combined action of UV-C and ozone [43−, B(OH)4−, and Cl−) on silicon isotope values still need to be investigated.More recent development of multicollector inductively plasma mass spectrometry (MC-ICP-MS) opens a variety of applications in geological, environmental, and material science where both high spatial resolution and accuracy are required . The dig14‰ (2σ) ,72. Meannd ozone ,74. The 14N16O polyatomic ions), the ion optics are operated in medium mass resolution mode, with which an external reproducibility better than ±0.23‰ (2σ) can be obtained. It is suitable to investigate the Si isotope signature of rock weathering at the micro-scale.An analytical protocol for accurate in situ Si isotope analysis has been established on a new second-generation custom-built femtosecond laser ablation system. The laser is coupled to a multicollector inductively coupled plasma mass spectrometer (fsLA-MC-ICP-MS) ,76. To r30Si) relative to the NBS-28 reference standard in the bulk silicate Earth, bulk Moon, and bulk silicate Mars are −0.29 ± 0.07‰, −0.27 ± 0.04‰, and −0.49 ± 0.03‰. [30Si in continental crust shows a narrow range from −0.43‰ to −0.15‰ with the relationship of δ30Si = 0.0056 × SiO2 (wt%) −0.567 [30Si of −0.38‰ to −0.27‰ with an average value of −0.32 ± 0.06‰ (2SD) for the altered oceanic crust is given by Yu et al. [30Si variations in various geological processes in terrestrial reservoirs is given in The silicon isotope compositions (δ± 0.03‰. . The δ30) −0.567 , and tha) −0.567 . The δ30u et al. , in goodu et al. . The genVISi and IVSi in majorite. In olivine polymorphs, olivine is slightly enriched in the heavy Si isotope compared to wadsleyite and ringwoodite due to their different crystal structures [http:// rruff.geo.arizona.edu/AMS/amcsd.php).Despite silicon being the defining element of silicate reservoirs on Earth, there is still no clear understanding of how much is hosted in Earth’s core or how the silicon-enriched continental crust forms from a long history of weathering, erosion, and subduction . Like thructures ,81 , the equilibrium silicon isotope fractionation properties of quartz (Q4), lizardite (Q3), kaolinite (Q3), and enstatite (Q2), and forsterite (Q0) were investigated by Méheut et al. (2009) [2Si3AlO10(OH)2) constituted of TOT layers , kaolinite (Al2Si2O5(OH)4) constituted of TO layers) have shown the similar silicon isotopic fractionation properties. The most recent understanding proposed that the silicon isotope fractionation properties among phyllosilicates  appear to be correlated with stoichiometry (Equation (5)) [eq = #Si, Aleq = 3/4#Al, and Mgeq = 1/2#Mg  are the charge-weighted stoichiometric coefficients of each cation, normalized to the charge of the silicon atom, and αX(T) are proportionality coefficients depending on temperature.The initial theoretical understanding of equilibrium silicon fractionation between quartz and kaolinite was given by Méheut et al. (2007) . With va. (2009) , which rion (5)) . It indiion (5)) .(5)10004SiO40 (H4), and its associated base H3SiO4− (H3) and H2SiO42− (H2), when the Si concentration is below the solubility limit of amorphous silica  [a1 = 9.84 and pKa2 = 13.2 at 298 K and 0 M ionic strength condition [4SiO40 and H3SiO4− species has been calculated using the first-principles methods based on ab initio molecular dynamics simulation, with which the fractionation factor (αH3-H4) of 0.9984 ± 0.0003  was obtained at 300K [H3-H4 values of 0.9963 ± 0.0009 and 0.9981 ± 0.0005 are derived from the isolated H4SiO4 and H3SiO4− species and hydrated species H4SiO4⋅(H2O)m and H3SiO4−⋅(H2O)n . Clearly, the consistence in αH3-H4 value of 0.9981–0.9984 from hydrated species from individual studies [2SiO42− (H2) is less than 0.59% when solution pH ≤ 11 (30Si of H4 and H3 species relative to the bulk solution (δ30Si = 0) with αH3-H4 values of 0.9963, 0.9972, 0.9981, and 0.9984 as a function of pH are estimated in The most common species of Si in solution at ambient conditions are silicic acid Hd pH ~8) . The fraondition . The equ at 300K , as show at 300K . The sil at 300K . With th at 300K , the αH3 studies ,87 verif pH ≤ 11 a, the δ32O molecules during cyclic freezing or evaporation [3+ and H4SiO40 on a stability diagram was provided using speciation calculation, in which a positive relationship between the occurrence of PS and the saturation degree was identified, suggesting polymerization would be precursors for the formation of secondary Al-silicates [4SiO4 + H4SiO4 = H6Si2O7 + H2O, etc.) and subsequently through growth of the nucleation of colloidal amorphous silica particles via continuing addition of silicic acid to the existing surfaces n = (SiO2)n-O-Si(OH)3 + H2O) [-, Na+, and Al3+, also have an impact on silica precipitation and phase transformation [B and KW are the thermal equilibrium constant for the dissociation of aqueous silica (Equation (6)) and the dissociation of H2O; cK′ is the silica solubility in water ; γ is the activity coefficient of silicate species H3SiO4−; S is the total solubility of silica under different pH and T conditions.In surficial geological processes, the occurrence of polymerized silicic acid (PS) might be caused by the weathering of silicate liberating from Al–Si-containing solids and polymerization of silicic acids in soil-liberating Hporation , which hilicates . In mariilicates . Generalilicates . In flui3 + H2O) . In addiormation ,97,98,99ormation ,99,100. ormation , it was ormation ). Using ormation ).(6)H4S4SiO4 and kaolinite–H4SiO4 were calculated to be αquartz-H4 of 1.0021  and αkaolinite-H4 of 1.0004  [precipitated-dissolved range from 0.9990 to 0.9996 at room temperature ranges, which confirms the preferential deposition of 28Si during abiotic silica precipitation [As shown in  ± 0.2‰)   . As showpitation ,103. Simpitation , Ca-isotpitation , and Mg-pitation .4SiO4) in solution, and the bond of 28Si–O is weaker than the bond of 30Si–O. The light silicon isotope is always preferentially taken up in polymerization and precipitation, which leads to dissolved silicon including less 28Si than precipitated silicon [30Si value of +1.1‰ in S-chert is almost identical to that of ~ +1‰ in Archean seawater [Precipitation of silica is a polymerization process of monosilicate  hydrolysis show that silica substitutes for double-corner, likely by forming 2C-type (double corner) complexes with small Fe oxy-hydroxide polymers whose structure consists of FeO6-octahedra linked together by common edges [mineral-solution of 0.9990  and 0.9984  for ferrihydrite and goethite, respectively [4SiO40 on ferrihydrite [3SiO4− (H3) over H4SiO40 (H4) species with reference to silicon isotope fractionation between H4 and H3  climate condition has indirect effects on soil δ30Si along a gradient of basalt-derived soils with the annual rainfall of 160–2500 mm, and bulk soil δ30Si declined in drier climate and increased in wetter climate soils with Si depletion; (iii) more negative δ30Si values in upper soil horizons are likely the result of additional fractionation that occurs during repeated dissolution and precipitation of secondary phases, while δ30Si values of soil horizons >1 m deep were better correlated with Si depletion [During chemical weathering processes (Reaction I), silicon is either being released into continental surface and ground waters, or transported into soils by uptake of plants, and formation of secondary precipitates , as well as adsorption onto secondary oxides. The associated dynamic fractionation occurs in precipitation during the weathering of silicate minerals, where the lighter silicon isotopes combines with Al and Fe-oxides to form the clay precipitation and the heavy silicon isotopes enrich in fluid phases or in secondary precipitation ,71. The  complex ,114, acc complex ,114,115.on edges . The silectively . On the ihydrite , we illu4 and H3 , which mFe-oxide . This reFe-oxide . As the Fe-oxide ,50,116. -cycling . The assepletion . Reactio30Si values in bulk soil and clay fractions in three weathering sequences at Guadeloupe formed under contrastive climatic conditions were compared by Opfergelt et al. [30Si in secondary clay minerals, such as slightly lighter δ30Si in smectite (−0.16 to −0.52‰) [30Si  in silicate rocks (BSE) [With the prevalence of silicon isotope as a useful weathering proxy, it raised the question of the role of mineralogy on silicon isotope fractionation by silicon incorporation into secondary phases. It is commonly accepted that the Si content in soils decreases progressively with increasing degree of weathering following preferential incorporation of light isotopes in secondary clay minerals , wht et al. . This stt et al. . It expl −0.52‰)  and very −0.52‰) ,117, comks (BSE) ,120.30Si , anferences ,118,119)wnstream . The assSilicon is involved in biological processes via the uptake of silicic acid by plant tissues and formation of deposits of amorphous silica , from which it can be released into soils and fluids by physical and chemical weathering and re-deposited in soils as secondary silicate minerals such as allophanes and imogolites or be exported to aquatic ecosystems ,123. The30Si values in plants [The role of complexation in biological fractionations of silicon isotopes is very poorly understood, largely because the complicated biochemical pathways, likely involving several enzymatic processes and active transport proteins, are not well constrained in any siliceous organism. Further, it was found that silicon isotope fractionations between plants and growth solutions were basically similar in both direction and extent for rice, bamboo, banana, and diatoms. It implied that some common and basic mechanisms, other than genes, might play important roles in the silicon uptake process for a wide range of plants . In genen plants ,21,22,23n plants ,78, whern plants ,102. Thef is the fraction of silicic acid remaining in solution, and δ30SiDSi and δ30SiDSio are the isotopic values of dissolved silicic acid at f and f =1, respectively.This fractionation factor can also be expressed as ε*; the per-mil enrichment factor between substrate and product may also be approximated as:30Si values and fractions of dissolved H4SiO4 from roots to stem and leaves [4 [4 [In addition to the Rayleigh effect in the dynamic fractionation of silicon isotope during the biological uptake into various plants, other controlling factors should be considered to explain obvious differences in both δd leaves ,23. As sd leaves ,20, it wd leaves : (i) Sild leaves , with a d leaves , at a tieaves [4 ; (ii) dies [4 [4 . The moses [4 [4 .30Si and decreasing dissolvable Si (DSi) in the oceans. In a longer temporal scale, major geological ) and biological events  impacted on the global oceanic Si cycle. As shown in 30Si over geological time was derived from the recorded δ30Si in chert, radiolarian, and sponge spicules, diatoms, which shows a rising trend compared to the Archean ocean generally. Meanwhile, during much of the Archean, the banded iron formations (BIFs) that contain, on average, 30% iron and 40% or more Si were most deposited prior to the initial rise of atmospheric oxygen at ~2.45 Ga in marine basins in stratified water columns [As discussed above, both biotic and abiotic silicification enriches light silicon isotopes, lifting δ columns , which i columns , which c4SiO40 concentration of the surface is the lowest and increases from the surface to 1000 m  [In a spatial scale, both the DSi concentration and the silicon isotope values in a local ocean are also variable with the depth increasing in water columns, as shown in o 1000 m a [24,25, ± 0.2‰)  and cent ± 0.2‰) , suggest30Si could help us evaluate the silicon budgets in terrestrial aqueous systems. After the pioneering works on the δ30Si of dissolved silicic acid in oceans, rivers, and estuaries [30Si distributions in various fluids have been clarified, such as freshwater  [30Si in summer than winter, while higher weathering degree releases lighter silicon isotopes into surface waters from silicate minerals. Compared to surface waters, groundwater with lighter δ30Si values possibly is the result of secondary clay minerals and silcrete dissolution.In addition to marine system, the variation of δstuaries , the δ30o +3.4‰) ,150,151,o +3.4‰) ,140,152.30Si values of −0.31 ± 0.07‰ [30Si value of −0.45 ± 0.05‰ for the lunar breccia 14304 [30Si values of −0.29± 0.06‰ for Low Ti basalt, 0.32± 0.09‰ for High Ti basalt, −0.29± 0.05‰ for lunar glass, and −0.27± 0.10‰ for highland rocks, and the data are in agreement with other isotope systems showing the majority of core formation happened early and before the giant impact [The origin of the Moon is still debated. The similarity (or not) of the Moon to the Earth in terms of major element budgets and the heterogeneity degree of lunar mantle provide key information as regards the composition of the Moon-forming impact and/or the processes operating during lunar formation. There are a few reported high-precision silicon isotope compositions in lunar, such as the average δ ± 0.07‰  and −0.3ia 14304 . More in30Si data relative to NBS–28 for samples from planetary bodies in the inner solar system concluded that the lunar mantle and BSE have similar δ30Si, and are significantly heavier than that in Mars, 4–Vestra and chondrite parent bodies ,,153,30Sif is the fraction of Si in the silicate phase; ε is the fractionation factor of Si isotope between metal and silicate; MBSE and MCore are the mass fractions of BSE and the core; c is the measured Si fraction in BSE, which is identified as 0.212 [It has long been proposed that the Earth’s core must contain a significant percentage of light elements to fit the density inferred from seismic studies. The light elements of hydrogen, carbon, oxygen, silicon, and sulfur are being considered . Accordias 0.212 .30SiBSE–meteorite of 0.15 ± 0.10‰, where the heavier δ30Si in BSE implied that Si partitioned into the metal phase during metal–silicate equilibrium at the time of core formation and the Δ30SiBSE–meteorite value indicated that the silicon content is from at least 2.5 wt.% to 16.8 wt.% in the Earth’s core when fixing the temperature of core formation to the peridotite liquidus [30Sisilicate–metal = 7.64 × 106/T2 in enstatite chondrites was measured by Ziegler et al. [30SiBSE–chondrite as the functions of ∆IW  and P(T) along magma ocean adiabat using the thermodynamics of Si solubility in metal established experimentally [30Si in metal free component of EH, EL, and carbonaceous/ordinary chondrite indicated that the Si isotope variation in the nebular gas did not induce the light Si isotope enrichment in enstatite chondrites [30SiBSE–BE because disagreement about the typical 30Si/28Si of chondrites and the inevitable uncertainty as to whether bulk Earth is truly chondritic in silicon isotope rather than the fractionation factor.The theoretical calculations provided the isotope fractionation factors of silicon (ε) is in the range of 0.4‰ to 2.0‰ in partitioning experiments ,163. Comliquidus . Relativliquidus , 6 wt.% liquidus ,54, and r et al. , which imentally ,164. A condrites . The driondrites . Recentlondrites . The lim30Si which has been interpreted as a hydrothermal signal [30Si values in Precambrian chert may reflect elevated temperature of the seawater, the influence of a continental source, or precipitation from isotopically heavy seawater [2O3 contents and rare earth element patterns [30Si values from +0.1‰ to +1.1‰, linking to the silicification of volcanic sedimentary precursor rocks [30Si-values reported for C-chert, which are often considerably lower than the value of −0.3‰ in modern deep-sea hydrothermal fluids, assuming that this was similar in Precambrian times [2O3 content showed obvious 30Si depleted compositions, e.g., down to −3.7‰ for Eoarchean banded iron formations from Isua, Greenland [Banded iron formations (BIFs) are chemical marine sediments that formed periodically throughout the Precambrian (3.8–0.5 Ga) and are usually characterized by alternating Fe- and Si-rich layers. The peak in BIFs formation between 2.5 and 2.3 Ga appears to correlate with major changes in the Earth’s history such as the rise of atmospheric oxygen and the change from anoxic to oxic conditions in the ocean . It has l signal ,167,168.seawater ,169,170.patterns ,110,170,or rocks ,110,169.an times . The C-creenland , −2.4‰ freenland ,171, −2.reenland ,172,173,reenland .30Si from −1.0‰ to −2.6‰ in bulk layers suggested rapid precipitation of the silicate phases from hydrothermal-rich waters, and the changes of Fe and Si isotope signatures directly reflect the upwelling dynamics of hydrothermal-rich water, which govern the rates of Fe and Si precipitation and the development of layering [30Si than coexisting translucent counterparts at the same stratigraphic level was interpreted as a primary feature acquired during deposition upon interaction between submarine discharging hydrothermal water and a stratified water body [A co-variation of Fe and Si isotope compositions was observed in a magnetite–carbonate–chert BIFs from the Archean Old Wanderer Formation in the Shurugwi Greenstone Belt, which was interpreted as largely primary signatures . The siglayering . The comter body .30Si vs. Al2O3%. Clearly, the C-chert with negative δ30Si and lower Al2O3 contents (less than 0.2%) indicates the non–equilibrium condition during rapid chemical precipitation caused by oversaturated fluid mixtures at relatively low ambient seawater temperature [30Si characters and the correlation of δ30Si and Al2O3 content implies that the relative contribution from isotope effect within silicification and precursor rocks with negative δ30Si values (−0.29 ± 0.08‰) [The metallogenic models of two typical chert deposits  are schematically shown in perature  during u± 0.08‰) . Those u18O and δ11B) can be used to better constrain the genesis of ore deposits. The origin of polymetallic ore deposits  and the Sullivan Pb–Zn ore deposit  have been well clarified. The Dachang Sn-polymetallic ore deposit  is one of the largest Sn deposits in the world, and the second largest Sn producer in China (more than 106 tons of tin) [30Si–δ18O values constrained the two individual origins of siliceous rocks: (i) Shallow radiolarian siliceous rocks without ore deposits. It is widely distributed with larger thickness and exhibits higher δ30Si (+0.2 ~ +0.8‰) and wider variation of δ18O values (+12.0 to +21.4‰). There is no direct relationship between the radiolaria siliceous rocks and mineralization, but the distribution along the Devonian deepwater rift trough might indicate the possible submarine exhalative environment. The existence of large amount of radiolaria in these rocks further approved the biochemical deposition genesis. (ii) The thin-laminated siliceous and K-feldspar rocks associated with cassiterite-sulfide ores. The negative δ30Si  and uniform δ18O values  mostly favored a submarine hydrothermal origin rather than the Yanshannian magmatic–hydrothermal origin [Silicon isotopic measurements used in combination with other geochemical indices (e.g δ of tin) . There a of tin) . A systel origin ,174. Thel origin  but are l origin .30Si values of 0 to +0.1‰, similar to that obtained from other clastic sediments [The Sullivan Pb–Zn ore deposit  is a classic example of stratiform sediment-hosted Pb–Zn deposit, and silicate minerals associated with the formation and post-depositional evolution of the orebody are widespread throughout the deposit . The claediments ,31,32. Tediments , which iediments .4). A frontier study was to track the source of secondary clay minerals  using silicon isotope composition [30Si survey on the secondary minerals from Southeast China, USA, and Italy indicated that the δ30Si in clay minerals varied in a wide range from −1.9 to +0.1‰ . Clay minerals from different sources can be distinguished by the δ30Si characters  in kaolinite in weathered origin is observed, and the δ30Si values shift negatively with increasing of weathering degree, which reflect a significant isotope fractionation during weathering, as discussed in 30Si values of −1.2 to −0.1‰ are between hydrothermal kaolinite and weathering kaolinite, perhaps showing mixed origins. Therefore, it is possible to constrain the origins of clay minerals in accordance using the δ30Si characters [The source and transformations of silicon in global biogeochemical cycles are complex, particularly in terrestrial settings where it can occur in numerous forms: Primary (unweathered) minerals, secondary crystalline minerals and non–crystalline forms, biogenic silica (phytoliths), and aqueous phases (Si(OH)position . A compaaracters . Kaoliniaracters .In this work, the fundamental advances in silicon isotope geochemistry investigations have been reviewed, including the silicon isotope fractionations linked to silicon complexations/coordination and thermodynamic conditions in various geological processes , and the geological implications for meteorites and planetary core formation, ore deposits formation and hydrothermal fluids activities, weathering rate, and silicon cycling, etc. On the basis of the distribution of silicon isotope compositions and the isotope fractionations associated with silicon transportation and cycling among different geological processes, the following issues could be concluded:4SiO40 on soil Fe–oxides/Al–oxides, and the degree of soil desilication; (iv) in the biological uptake processes, biological fractionations of silicon isotopes are very poorly understood, largely because the complicated biochemical pathways, likely involving several enzymatic processes and active transport proteins.The silicon isotope fractionations in various geological processes are distinctive: (i) In the high-temperature process, the equilibrium silicon isotope fractionation during the mantle–core differentiation results in the observed heavy isotope composition of the bulk silicate Earth (BSE) if the Earth accreted only from chondritic materials; in low-temperature precipitation, it is strongly dependent on the degree of (metastable) equilibrium in the silica–water system  and the presence of carrier phase ; (ii) the equilibrium fractionation of silicon isotopes among silicate minerals are sensitive to the Si–O bond length, Si coordination numbers (CN), the polymerization degrees of silicate units, and the electronegativity of cations in minerals. (iii) In chemical weathering processes, the isotope fractionation is controlled by the associated dynamic fractionation in precipitation during the weathering of silicate minerals, the selective adsorption of H30Si values in various lunar materials  and BSE approved the majority of lunar materials formed early and before the giant impact. (ii) The equilibrium silicon isotope fractionation during the mantle–core differentiation provided the potential to evaluate the Si-core concentration and to explore the driving force for the light Si isotope enrichment in enstatite chondrites. (iii) The combination of δ30Si and other geochemical indices would well constrain the genesis of ore deposits and the hydrothermal fluids activities. For instance, the metallogenic models of two typical chert deposits  could be identified in accordance with the distribution pattern of δ30Si vs. Al2O3%. The origin of polymetallic ore deposits  has been well clarified with Si–O isotopes in siliceous rocks, Sr–Nd isotope systematics of tourmalines, and chondrite–normalized rare earth elements (REE) patterns. The δ30Si has the potential to be a useful weathering proxy, tracking the origins of clay minerals and desilication degree of soils.With further understanding on the driving mechanisms for silicon isotope fractionations behind typical geological processes, the silicon isotope geochemistry provides powerful geochemical constraints for tracing bio-physico-chemical processes in terrestrial environments, weathering processes, mineral deposits formation, hydrothermal fluids activities, and meteorite and planetary evolutions, etc. (i) The comparison on δIn the future work, it is urgent to provide rigorous experimental and/or theoretical studies to calibrate critical effects on isotopic fractionation during melting, core formation, and differentiation, and to better understand the mechanism of dynamic isotope fractionation of silicon during silicon cycling from lithosphere to biosphere and transportation in mineral digenesis.