Interestingly, esr2a−/− females showed apparently normal sexual behavior but without oviposition in response to male courtship. This phenotype indicates that physical readiness and motivation of sexual behavior is independently controlled.In vertebrates, sex steroids play crucial roles in multiple systems related to reproduction. In females, estrogens and their receptor estrogen receptor (ER or Esr) play indispensable roles in the negative sex steroid feedback regulation of pituitary gonadotropin secretion, which prevents excessive development of ovarian follicles. However, the mechanism of this feedback regulation of a gonadotropin, follicle stimulating hormone (FSH), which is essential for folliculogenesis throughout vertebrates, is poorly understood. In the present study, we generated knockouts of all subtypes of nuclear estrogen receptors in a model teleost medaka, which is suitable for the study of endocrine control and behavioral assays, and analyzed fertility, behavior and functionality of estrogen feedback in each knockout line. Among the estrogen receptors, we revealed that an estrogen receptor Esr2a plays an essential role in this feedback regulation. In addition to this, we also found that  In female vertebrates, a group of sex steroid hormones, estrogens, which are mainly produced by ovarian granulosa cells, are suggested to play crucial roles in multiple systemsOne of the most important roles of estrogens is the feedback regulation of gonadotropin (follicle stimulating hormone (FSH) and luteinizing hormone (LH)) secretion from the pituitary. During the folliculogenesis in female vertebrates, estrogens released from mature gonad suppress the secretion of gonadotropins from pituitary in a negative feedback manner, to prevent excessive development of ovarian follicles. In addition to such neuroendocrine regulation, estrogens are suggested to be involved in a wide variety of sex-related phenomenons, such as sexual differentiation, sexual behavior, and development of female-specific organs.7. In vertebrates, two subtypes of ER, Esr1, also referred as ERα, and Esr2, also referred as ERβ10, have been generally conserved13.Majority of estrogenic actions are suggested to be mediated by nuclear estrogen receptors (ERs), which belong to the steroid hormone receptor family15. However, growing body of evidence strongly suggests that the kisspeptin-mediated gonadotropin release can be a mammalian-specific mechanism18. Therefore, the role of estrogen receptors in the steroid feedback in mammals cannot be applied to that of vertebrates in general, and the general mechanisms of steroid feedback regulation by estrogens in vertebrates are still unknown.In mammals, it has been strongly suggested that Esr1 expressed in hypothalamic kisspeptin neurons play essential roles in this feedback regulation of folliculogenesis by changing GnRH release from hypophysiotropic GnRH neurons and pulsatile release of LH from the pituitary19. On the other hand, the sex is strictly determined by the sex determination gene, dmy in a teleost, medaka (Oryzias latipes)20, which led us to successfully analyze the contribution of each ER subtype to the female reproductive functions. It has been reported that teleosts possess three ERs. Esr1 and Esr2 were duplicated in the early gnathostome lineage, and Esr2 was further duplicated in the third-round whole genome duplication event in teleosts. Esr2a and Esr2b are considered to share roles of their common ancestor, Esr2, after this sub-functionalization23.A previous study has challenged to clarify the function of ERs in a model teleost zebrafish by knockout (KO) technology. However, because of their sexual plasticity, ER-KO zebrafish easily underwent sex change to males, and female functions could not be analyzedIn the present study, we established ER KO lines for each ER subtype by using CRISPR/Cas9 and analyzed their phenotypes from several viewpoints such as fertility, ovarian morphology, and expression of gonadotropin genes in females of each ER KO line. From the analyses of the established line for each ER, we here provide the first experimental evidence to show that Esr2a expressed in the pituitary plays an essential role in the regulation of female reproduction, while the other two subtypes of ERs, Esr1 and Esr2b, are dispensable for that. Moreover, we also found the essential role of Esr2a in the oviduct formation, dysfunction of which results in complete infertility.24, and we confirmed that each ER has lost its functionality. According to the fact that all homozygotic males were fertile, we analyzed only KO females of each ER KO in the following analyses. In the present study, we used offsprings of homozygous males and heterozygous females, which includes theoretically the same number of homozygotes and heterozygotes with non-biased backgrounds in a single tank.The designed CRISPR guide RNAs successfully cleaved targeted sites of each gene and induced frameshifts for each gene. After incross and/or outcross with wild type (WT), sequence analysis was performed in the targeted genes of each ER KO medaka , and homo (esr−/−) KO female medaka of each ER subtype to analyze their fertility. The mean number of eggs spawned by each genotype during analyzed six days was as follows: WT, 16.0 ± 1.4; esr1+/−, 7.1 ± 2.3; esr1−/−, 7 ± 1.4; esr2a+/−, 11.6 ± 2.1; esr2a−/−, 0; esr2b+/−, 10.3 ± 2.7; esr2b−/−, 4.61 ± 2.3 , while the others could spawn more or less normally. In agreement with the description in a recent review article25, some of the adult females of esr2a−/− showed abnormal bodily swelling. This abnormal swelling is probably because of the failure in oviposition due to the defective oviduct formation, which caused the accumulation of eggs.We counted the number of eggs spawned by WT, hetero . For further analysis of the phenotype of each KO line, the ovaries of ER KO medaka were sectioned and were histologically examined by hematoxylin and eosin (HE) staining . Thus, all three subtypes of ERs were shown to be dispensable for folliculogenesis.Next, we analyzed gonadosomatic index (GSI) to assess the maturity of the ovaries. There was no significant difference among them , 00:05-00:07; Supplementary Movie esr2a+/−), immediately after the start of Movie esr2a−/− females showed all of those steps of normal courtship behavior, they failed to perform oviposition  expression in the pituitary of KO females in each ER subtype, we performed quantitative reverse transcription PCR (qRT-PCR). Esr2a−/− females showed significantly higher fshb expression than the others except esr1−/−  is the most abundantly produced and shows the strongest estrogenic effect on gonadotropin expression. In the previous study using medaka that examined the effects of E2, it was reported that fshb by E2, we examined E2 effects on the fshb expression by using ovariectomy (OVX) and OVX + E2 model in esr2a+/− and esr2a−/−. OVX + E2 medaka of esr2a+/− showed significant decrease in the expression of fshb when compared with that of sham operated (sham) or OVX medaka in a similar manner to WT26  of isolated pituitary. After exposure to 10 nM E2 for 20 hours (h), fshb expression was significantly decreased to the extent comparable to OVX + E2 treatment , which spawn every day, showed oviduct with complete opening  Fig. . The perzed Fig. . For comage Fig. . Among tary Fig.  does notesr2a−/− is due to the oviduct atresia. Eventually, older females (age: six to eight months) of esr2a−/− showed complete atresia of oviduct  plays crucial roles in estrogen-dependent GnRH/LH secretion because kisspeptin neurons expressing ESR1 in the arcuate nucleus (ARC) mediate negative feedback regulation by estrogensesr2a−/− adult females showed atretic oviduct  at a water temperature of 27 °C. The fish were fed two to four times a day with live brine shrimp and flake food. All experiments were conducted in accordance with the protocols approved by the animal care and use committee of Graduate School of Science, the University of Tokyo .esr1 was transcribed by using a gRNA expression vector constructed from DR274 . CRISPR RNA and tracer RNA for esr2a or esr2b were synthesized by a commercial company . The injected embryos were intercrossed for the identification of the germ line founders with mutations in each target locus. Their offsprings were then outcrossed for the identification of the individual founder fish. Heterozygous transgene carriers in the F1 generation were identified by the melting curve and/or sequence analyses using primers described in the primer list  was injected into the cytoplasm of one- or two-cell-stage embryos (F0). Guide RNA (gRNA) for For the analysis of GSI of each KO line (age: three to four months), we used females that had been separated from males from the previous night of the sacrifice to avoid egg release (spawning). After confirming that they did not spawn, we dissected the ovary and measured the ovarian weight at noon. The GSI was calculated as the ovary weight/body weight × 100 (%).esr2a+/− females that held ovulated eggs in their ovary could spawn, and their GSI must be decreased for that.To analyze the sexual maturity of females that were used in quantitative PCR (qPCR) experiment, we calculate their GSI as shown above. We used females that had not been separated from males from the previous night of the sacrifice. Thus, esr1+/−, esr1−/−, esr2a+/−, esr2a−/−, esr2b+/−, esr2b−/− were mated with WT male medaka as one to one male-female pair (Day 1). After habituation, the number of eggs spawned was counted from Day 17 to Day 22.Female medaka (age: three to four months) of WT and Female medaka (age: three to four months) were deeply anesthetized by MS-222, and ovary samples were then treated with a Bouin’s fixative at room temperature for one to three days. After fixation, each sample was dehydrated with ethanol or methanol, cleared with xylene and embedded in paraffin. Sections of 8-9μm thickness were stained with hematoxylin and eosin (HE). The sections were observed under an upright microscope . Photographs were taken with a digital camera .esr2a+/− and esr2a−/− were mated with sexually mature male medaka one by one. Movies were taken using raspberry pi and raspberry pi camera V2  from ten minutes before light on to one hour after light on time, during which spawning of medaka usually occurred. We analyzed the sexual behavior for nine days.Four to five months old female medaka of esr2a+/− and esr2a−/− (age: three to four months) were deeply anesthetized and their pituitaries were collected for qRT-PCR.Female medaka of TM RT Master Mix (TaKaRa). For qRT-PCR, the cDNA was amplified by KAPA SYBR FAST qPCR kit  with AriaMX Realtime PCR System . The temperature profile of the reaction was 95 °C for 3 minutes, 40 cycles of denaturation at 95 °C for 10 seconds, annealing at 60 °C for 10 seconds, and extension at 72 °C for 10 seconds. The PCR products were verified using melting curve analysis. The data were normalized by a housekeeping gene, ribosomal protein s13 (rps13). The melting curve analyses were conducted to ensure that the amplicons were the same as the sequence-certified ones. The primer pairs used in the real-time PCR are listed in the primer list  according to the manufacture’s instruction. Total RNA samples were then reverse transcribed by PrimeScriptAdult female medaka of WT (age: three to four months) were deeply anesthetized and their brains, pituitaries, and oviducts were collected for reverse transcription PCR (RT-PCR) analysis. We pooled each tissue from three individuals.TM RNA Basic Kit (Nippon Genetics) according to the manufacture’s instruction. RNA samples (20 ng) were then reverse transcribed by PrimeScriptTM RT Master Mix (TaKaRa). For RT-PCR analysis, the cDNA was amplified by KAPA HiFi HotStart ReadyMix (Nippon Genetics) with T100TM Thermal Cycler . The temperature profile of the reaction was 98 °C for 1 minute, 20 cycles of denaturation at 98 °C for 10 seconds, annealing at 70 °C to 60 °C (−0.5 °C/cycle) for 5 seconds, and extension at 72 °C for 1 minute followed by second amplification step of 15 cycles of denaturation at 98 °C for 10 seconds, annealing at 60 °C for 5 seconds, and extension at 72 °C for 1 minute. The PCR products were separated on 2% agarose gels, and we verified that the amplicons matched the predicted sequence of esr2a cDNA by sequence analysis. The primer pairs used in the PCR are listed in the primer list  and their ovaries were excised via intraperitoneal operation. The OVX medaka were allowed to survive for at least four days. One group of the OVX medaka were fed with E2-  (Sigma-Aldrich) containing food in addition to the normal food for three days. After the steroid treatment, the medaka were anesthetized, and their pituitaries were collected for RT-qPCR analysis.Ovariectomy was performed as previously described42, we expect that 10 nM E2 would be a reasonable physiological concentration for a female medaka. After incubation, the pituitaries were homogenized and extracted RNA for qPCR. The total RNA extraction and qPCR were performed as described above.Female medaka of WT (age: six to seven months) were deeply anesthetized and decapitated. The pituitary was collected and incubated in the medium with or without 10 nM E2 at 25 °C for 4 hours (h), 12 h, or 20 h. We prepared the medium as follow;  supplemented with L-glutamine (2 mM), D-glucose (10 mM), penicillin-streptomycin , and 5% fetal bovine serum . Given the data available for previous studiesesr2a+/− and esr2a−/− (age: three to four months) were deeply anesthetized and decapitated. The pituitary was collected and incubated in the medium with or without 10 nM E2 at 25 °C for 20 h. After incubation, the pituitaries were homogenized and extracted RNA for RT-qPCR as same method described above.Female medaka of Female medaka were deeply anesthetized by MS-222, and whole bodies samples were then treated with a decalcifying solution containing 10% formic acid and 70% ethanol or methanol. Then, decalcified whole bodies were dehydrated with ethanol or methanol, cleared with xylene and embedded in paraffin. Sections of 8-9μm thickness were stained with HE. The sections were observed under an upright microscope . Photographs were taken with a digital camera .esr2a+/−, and esr2a−/− female medaka were mated with WT male medaka as one or two females to one male. After habituation, we observed their ovarian morphology and took pictures with a digital camera  attached to a stereoscopic microscope .WT, fshb or lhb were analyzed by Tukey-Kramer test or t-test.The number of spawned eggs, and GSI of WT and ER KO were analyzed by Steel’s test, a nonparametric multiple comparison with WT control. The qPCR data were expressed as mean ± SEM. The expression levels of Supplementary informationSupplementary movie1 esr2a homoKOSupplementary movie2 esr2a heteroKO


Direct (utilize easily available and abundant precursors) and selective (both chemo- and regio-) aliphatic C–H functionalization is an attractive mean with which to streamline chemical synthesis. With many possible sites of reaction, traditional methods often need an adjacent polar directing group nearby to achieve high regio- and chemoselectivity and are often restricted to a single site of functionalization. Here we report a remote aliphatic C–H thiolation process with predictable and switchable regioselectivity through NiH-catalysed migratory hydrothiolation of two feedstock chemicals . This mild reaction avoids the preparation of electrophilic thiolation reagents and is highly selective to thiols over other nucleophilic groups, such as alcohols, acids, amines, and amides. Mechanistic studies show that the reaction occurs through the formation of an RS-Bpin intermediate, and THF as the solvent plays an important role in the regeneration of NiH species. Construction of C–S bonds via C–H functionalization is an attractive route to organosulfur compounds. Here, the authors show the synergistic combination of NiH-catalysed alkene isomerization and subsequent thiolation to afford remote hydrothiolation products from alkenes  and thiols under mild conditions. They compose ~20% of all Food and Drug Administration-approved drugs5. The development of protocols for the sustainable and efficient construction of C–S bonds is important in chemical synthesis. Commonly used methods for the construction of such bonds include Michael addition, SN2-type alkylation, and the powerful transition-metal-catalyzed C–S cross-coupling11. One potential and more attractive strategy for their construction is through the selective C−H functionalization9, because this leads to the utilization of more widely available starting materials or more concise synthetic routes. However, to achieve excellent regio- and chemoselectivity, most of these processes need a polar directing group in the vicinity, and this limits their application in organic synthesis. As an alternative, the recently emerging metal-hydride-catalyzed15 olefin remote functionalization53 can install a functional group at a distal position in a hydrocarbon chain under mild conditions. Starting from the ubiquitously available olefin-containing substrates, and using an extra hydride source, the NiH-catalyzed remote hydrofunctionalization53 with aryl/alkyl halides as electrophiles has been established as a powerful protocol for the construction of a diverse range of C–C bonds at a distal, inert sp3 C–H position  with benzyl mercaptan (2a). After careful evaluation of a variety of nickel sources, ligands, bases, hydride sources, and solvents  as a ligand, HBpin (pinacolborane) as the hydride source, Li3PO4 as the base, and mixed tetrahydrofuran/acetonitrile (THF/CH3CN) as the solvent delivers the desired migratory benzylic thiolation product (3a) in 75% isolated yield as a single regioisomer [regioisomeric ratio  >99:1]  leads to inferior yield and regioisomeric ratio  are found to perform well producing the desired benzylic thioether exclusively  and electron-deficient (3d–3f and 3 h) arenes are suitable for this reaction. Structurally complex aromatic systems such as sugar-linked aryl ring (3j) and camphor-linked aryl ring (3k) are amenable to the migratory cross-coupling. Heteroaromatic substrates, such as those containing a pyridine-linked aryl ring (3l) or a thiophene (3m) in place of the aryl group, are also well tolerated. Unactivated internal olefins also readily undergo alkene isomerization-hydrothiolation smoothly  react well, and high selectivity for thiolation at the benzylic position is observed, regardless of the starting position of the C=C bond. For substrates with a tertiary carbon on a benzyl position, which previous reports48 have noted as challenging, migration towards the benzylic position and subsequent thiolation to generate the S-containing tetrasubstituted carbon center is still preferred (3t). Styrenes themselves (3u–3c′) are also suitable partners under these conditions , a boronic acid pinacol ester (3v), an aryl nitrile (3w), and an ester (3c′). The reaction can also be extended to α-methyl styrenes to provide exclusively the benzylic thioethers (3b′ and 3c′) with a fully substituted carbon center.With the optimal conditions in hand, we sought to define the scope of the alkene component Fig. . First, ely Fig. . Substrahly Fig. . As experyl ring –3l are f3d′ and 3e′, Fig. 3f′ and 3g′, Fig. It is important to highlight that alkynes, another type of easily prepared starting material, could undergo reductive remote hydrothiolation to deliver the same migratory thiolation products (4b–4m) and aromatic (4n–4c′) thiols are excellent reaction partners and give the corresponding benzylic thioethers with good to excellent yields and regioselectivities. An array of primary and secondary aliphatic thiols all prove to be competent substrates, delivering the desired benzylic thiolation products in good to excellent yields (4b–4l). For the steric hindered tertiary thiol, a disulfide can be used to obtain a satisfactory yield (4m). In addition, a variety of electron-withdrawing (4o–4u and 4w) and electron-rich (4v and 4x–4b′) thiophenol derivatives are competent substrates. A variety of heterocycles frequently found in medicinally active agents, including both furan  and thiophene (4j), are also compatible, and a variety of functional groups are readily accommodated, including esters (4e and 4h), an aryl fluoride (4p), an aryl chloride (4q–4s), and ethers (4u–4x). Notably, potential coupling motifs, including a primary alcohol (4f), a primary carboxylic acid (4g), a phenol (4y), a primary aniline (4z), a secondary Boc carbamate (4h), and a secondary acetyl amide (4a′) remain intact, which demonstrates both the excellent chemoselectivity of this transformation and their potential application in selective cysteine conjugation in biomolecules.Further investigation of the reaction demonstrated the broad scope of thiol partner Fig. . In gene1a was subjected to the standard reaction conditions in the absence of any thiol. A significant amount of other olefin isomers arising from the olefin isomerization is observed within 1 h, which indicates that occurrence of olefin isomerization does not depend on the presence of the thiol and also suggests that olefin isomerization is unrelated to C–S coupling  is resubjected to the standard conditions, suggesting that chainwalking preceeds the C–S coupling , this migrated hydroboration intermediate was resubjected to the standard conditions. However, no desired thiolation product was observed, suggesting that the C–S coupling step does not proceed through the remote hydroboration intermediate , however, indicates that the disulfide (δ = −114.9 ppm) is first transformed into an RS-Bpin intermediate (δ = −116.8 ppm)  also reveal the generation of this RS-Bpin intermediate (δ = −116.8 ppm) with no trace of disulfide detected  analysis. Overall, these results reinforce the notion that the disulfide is not involved as the active intermediates from the thiol, and suggests that the in situ generated RS-Bpin might be the actual thiolation reagent.To shed light on the thiolation process, a variety of experiments were carried out. When 0.5 equiv. of a symmetrical disulfide is used instead of 1.0 equiv. of the corresponding thiol, the desired remote hydrothiolation product is obtained in a comparable yield Fig. , indicatpm) Fig. . Signifited Fig. . Meanwhi2A′, generated in situ from the thiol and HBpin, a competent yield of 3a is obtained. In this case, only a stoichiometric amount of the alkene is required and the desired thiolation product could still be obtained in comparable yield  when a slightly excess of alkene or RS-Bpin is used (1.2 equiv.). This finding, together with the results disclosed in Fig. Encouraged by these results, we wondered whether the pre-generated RS-Bpin reagent could be employed directly instead of a thiol. Indeed, as shown in Fig. d8 is used in both standard and modified standard reactions, a small amount of deuterium scrambling and deuterium incorporation at all positions except the benzylic position along the aliphatic chain of the desired product is observed by 2H NMR and mass spectrometric analysis. This indicates that a small amount of NiD is involved in the chainwalking process and the small amount of NiD should come from the deuterated THF-d8.Control experiments reveal that the solvent THF plays an important role. Only 5% yield of desired product is observed in the absence of this solvent. As shown in Fig. 11B NMR) experiments were carried out to trace the standard reaction. As shown in Fig. δ = 33.6 ppm) is confirmed again by 11B NMR spectroscopic analysis. We could also observe two new boron signals accompanied with the consumption of THF, which matches the signals of BpinOBpin (δ = 21.2 ppm) and ROBpin (δ = 22.3 ppm).To probe further the role of THF, Boron-11 NMR (I)66, which is initially formed from a Ni(II) precursor, a ligand, and an hydride source, inserts into the alkene (1a), and initiates the relatively fast and reversible chainwalking process through iterative β-hydride elimination/migratory reinsertion processes. A series of isomeric alkylnickel(I) species  is then accessed through this chainwalking process. Controlled by the choice of ligand, selective reaction of the benzylic alkylnickel(I) intermediate (IV) with the thiolation reagent, the RS-Bpin (2A′) generated in situ from thiol and pinacolborane, probably through an oxidative addition and sequential facile reductive elimination70 process then delivers the benzylic thiolation product (3a) along with LNi(I)Bpin (V). The active LNi(I)Bpin (V) species is then captured by THF to generate the corresponding alkylnickel(I) intermediate (VI). Sequential chainwalking and β-oxy elimination would deliver the isomeric mixture of butene along with LNi(I)OBpin (VII). The nickel hydride species (I) is then regenerated in situ by a stoichiometric amount of the pinacolborane to complete the catalytic cycle. Additional studies aimed at a full elucidation of the reaction pathway are in progress.Although an in-depth mechanistic discussion must await further investigation, a description of the proposed pathway, based on the above mechanistic studies, is shown in Fig. 3s and 3o) can be obtained in high yield as a single regioisomer in both cases .Finally, as shown in Fig. In summary, we have developed a NiH-catalyzed remote hydrothiolation reaction of alkenes using thiols directly as thiolation reagents. This transformation utilizes readily accessible alkenes/alkynes and thiols as starting materials and earth-abundant nickel salts as catalysts. The mild process allows the direct installation of a thioether group at a benzylic, α-ether, or a terminal position with excellent regio- and chemoselectivity, as well as high functional group tolerance. Moreover, mechanistic studies reveal that the activated thiolation reagent is the RS-Bpin intermediate, and the ether solvent plays an important role in the regeneration of NiH species. Finally, the practical value of this transformation is highlighted by the regioconvergent conversion of unrefined isomeric mixtures of alkenes. The application of this protocol in cysteine bioconjugation as well as an asymmetric version of the current transformation is currently in progress and will be reported in due course.2  and bathocuproine  . The vial was introduced into a nitrogen-filled glove box, anhydrous THF (0.40 mL) and CH3CN (0.20 mL) were added, and the mixture was stirred for 10 min, at which time alkene , benzyl mercaptan , HBpin  and Li3PO4  were added to the resulting mixture in this order. The tube was sealed with a teflon-lined screw cap, removed from the glove box and stirred at 60 °C for 24 h (the mixture was stirred at 750 rpm). After the reaction was complete, the reaction mixture was immediately filtered through a short pad of silica gel (using EtOAc in hexanes) to give the crude product. n-Tetradecane (20 μL) was added as an internal standard for GC analysis. 1,1,2,2-Tetrachloroethane  was added as internal standard for 1H NMR analysis of the crude material. The product was purified by chromatography on silica gel for each substrate. The yields reported are the average of at least two experiments, unless otherwise indicated. See Supplementary Information for more detailed experimental procedures and characterization data for all products.To an oven-dried 8 mL screw-cap vial equipped with a magnetic stir bar was added NiISupplementary InformationPeer Review File


These changes in Ca2+ release amount can reveal the mechanism underlying how the interaction between Ca2+ release and Ca2+ uptake induces Ca2+ alternans. This study of alternans by calculating the values of Ca2+ release properties with impaired SERCA has not been explored before. Here, we induced Ca2+ alternans by using an impaired SERCA pump under ischemic conditions. The results showed that the recruitment and refractoriness of the Ca2+ release increased as Ca2+ alternans occurred. This indicates triggering Ca waves. As the propagation of Ca waves is linked to the occurrence of Ca2+ alternans, the “threshold” for Ca waves reflects the key factor in Ca2+ alternans development, and it is still controversial nowadays. We proposed the ratio between the diastolic network SR (NSR) Ca content (Cansr) and the cytoplasmic Ca content (Cai) (Cansr/Cai) as the “threshold” of Ca waves and Ca2+ alternans. Diastolic Cansr, Cai, and their ratio were recorded at the onset of Ca2+ alternans. Compared with certain Cansr and Cai, the “threshold” of the ratio can better explain the comprehensive effects of the Ca2+ release and the Ca2+ uptake on Ca2+ alternans onset. In addition, these ratios are related with the function of SERCA pumps, which vary with different ischemic conditions. Thus, values of these ratios could be used to differentiate Ca2+ alternans from different ischemic cases. This agrees with some experimental results. Therefore, the certain value of diastolic Cansr/Cai can be the better “threshold” for Ca waves and Ca2+ alternans.Impaired sarcoplasmic reticulum (SR) calcium transport ATPase (SERCA) gives rise to Ca Meanwhi([ADP]i)  and pH i([ADP]i) . Accordi2+ alternans can occur or not in different ischemic conditions. We attempted to find the ranges of CL in which Ca2+ alternans could arise in these three cases. In our simulations, the starting CL is 250 ms and the increasing step is 10 ms. Finally, we determined the ranges of CL in ischemic cases 1, 2, and 3, which are from 250 ms to 280 ms, from 250 ms to 380 ms, and from 250 ms to 300 ms, respectively.The CL also affects whether Ca2+ ) comprised the amount of diastolic NSR Ca2+ and the junctional SR (JSR) Ca2+. The amount of Ca2+ release (Cak)release( (mmol)) was expressed as the integral of the Ca2+ release flux (Jk)rel( (mmol/L/ms)) on the kth beat (equation (k)release( to Cak−1)sr_total( to represent the fraction of SR Ca2+ release:2+ included free and buffered Ca2+ (Cajsr_free (mmol/L) and Cajsr_buff (mmol/L)); vnsr and vjsr represented the volume of NSR and JSR; and Cak)sr( (mmol/L) and Cak)nsr( (mmol/L), respectively, referred to the diastolic SR and NSR Ca2+ content on the kth beat.In our simulations, the total amount of diastolic SR Caequation ). Then, 2+ is released through CRUs. One CRU is set to have six neighbors in the 3D-distribution cell simulation [N0 represents the total number of CRUs and NK is the number of that activated on the kth beat [α represents the probability of a Ca spark being activated spontaneously or by the L-type Ca2+ current (ICaL); β is the probability of a Ca spark triggered on the kth beat being unavailable during the (k + 1)th beat; γ indicates the probability of a Ca spark recruiting one of its neighboring; and f represents the percentage of secondary Ca sparks in the remaining available CRUs [k + 1)th beat is given as follows [2+ depletion of each CRU and Casr is the average Ca2+ content of each CRU before release [b> refers to the average Ca2+ content of these Nk CRUs after they sparked [k)releaserelease( and Cak−1)sr_totalNK+1= release . <Cab> r sparked . We put equation . The lefquations ), were lNK and N0 in equation (k)releasesr_totalrelease( and Cak−1)sr_total was from zero to the maximum Casr_total/(vnsr + vjsr). Then, these values were input to solve equation amplitude more prominent, and leads to a slightly deeper notch of the AP. Subsequently, the voltage-dependent repolarization currents cause different repolarization phases.According to equation , increasinset of . APs alsinset of , due to 2+ alternans can be observed in all three ischemic cases when CL = 250 ms. However, when the CL increases to 300 ms, it can only be observed in cases 2 and 3. In case 1, the maximum Casr with CL of 300 ms does not reach the value of Casr at which bifurcations occur with CL of 250 ms. In addition, the slopes of curves change slightly before alternans onset  affects the ratios more effectively. Furthermore, how diastolic Casr, Cai, and their ratio change with sequent heartbeats is analyzed under transient Ca2+ alternans . This means the maximum diastolic Cansr/Cai can be affected by ischemic cases. In addition, the onset of alternans is induced by ischemia, and thus its “threshold” is taken to be different with ischemic degrees. In nsr/Cai differs with ischemic conditions. As a contrast, neither Cai nor Cansr is able to distinguish different ischemic cases. In 2+ alternans, Casr and Cai increase. Correspondingly, Iup goes on retaking the increasing released Ca2+ and diastolic Cansr/Cai keeps rising. However, to what extent Iup and diastolic Cansr/Cai can increase is limited by the thermodynamic constrains. Ca2+ alternans and Ca waves form when the unbalance between the Ca2+ uptake and Ca2+ release occurs. Subsequently, as diastolic Cai and Cansr keep on increasing, the Ca2+ uptake rate increases enough to uptake all released Ca2+ and Ca2+ alternans disappears. This final constant value of diastolic Cansr/Cai indicates the new balance between the Ca2+ release and Ca2+ uptake. On the other hand, if diastolic Cai or Cansr is the “threshold” for Ca waves and Ca2+ alternans, these two increasing values will initiate larger Ca waves and Ca2+ alternans will not disappear.SERCA pumps contribute to maintain the Caequation , the max2+ efflux cooperates with the influx to affect the “threshold” for alternans. An intermediate SR Ca2+ uptake rate and a larger SR Ca2+ release work synergistically to produce alternans at longer CLs [Iup contributes to unbalanced Iup and Irel and promotes abnormal intracellular Ca2+ handling. In addition, other studies demonstrated that the properties of RyRs affect the “threshold” of Casr for alternans [sr [2+ uptake rate becomes larger accordingly. As the physiological conditions  are identical, the maximum Ca2+ uptake rate is kept the same. Thus, maximum Iup occurs at a lower Casr, resulting in a reduced “threshold” of Casr for alternans (Xie et al.  demonstrnger CLs . Accordilternans , 41, 42.Irel during the formation of Ca2+ alternans. Our new “theory” shows that the unbalance between Iup and Irel begins at the “threshold” of diastolic Cansr/Cai. These two currents interact with each other and result in Ca2+ alternans. We should also note that Ca2+ alternans of case 3 is transient (Iup and Irel get balanced, Ca2+ alternans is suppressed. Therefore, this “threshold” theory may disclose a novel therapeutic strategy for Ca2+ alternans. In theory, keeping the ratio below the “threshold” stops Ca2+ alternans occurrence. In clinical, the treatment aiming at the interaction between these two currents may have a promising effect. Although direct therapeutic tools modulating the SR release channels have not been fully developed [This “threshold” theory provides a new idea for changes of ransient , which s2+ alternans. That is, these components of Ca cycling interact with each other to affect Ca2+ alternans development. In addition, compared with isolated changes of diastolic Casr and Cai, the value of diastolic Cansr/Cai is more appropriate to function as the “threshold” for alternans. By defining this new “threshold,” we can better explain how the interplay between the Iup and Irel causes alternans. Furthermore, this proposed “threshold” theory may help find therapeutic targets for suppressing Ca2+ alternans.The integrated cell model can be used to simulate the SERCA pump function in specific ischemic conditions, whereas the original ORd model cannot be used. The simulated results indicate that Ca waves can be induced by impaired SERCA pump and thus give rise to Cai chosen in our simulation can also determine the results by influencing the inputs. We also need to take use of other cell models to calculate values of these properties during Ca2+ alternans occurrence and identify our “threshold” theory. In addition, although we conclude that Iup and Irel interact to produce Ca2+ alternans, we just identify how the Ca release is affected by impaired SERCA pump, and the effect of Irel on Iup should also be identified in detail in the future.This model is just used to simulate the impaired SERCA pump function during ischemia, whereas L-type calcium and other currents are also affected under ischemia, and these changes are not included in our model. We need to further improve this integrated model to simulate more accurate ischemic conditions. It is noted that the values of the three properties can be different by using different groups of inputs. The range of Ca


We confirmed that passage times of whole blood with heparin + EDTA-2Na were always shorter than those of whole blood with only heparin in healthy subjects and patients with DM or ACS under suction pressures of − 30 cmH2O. There was a significant correlation between delta whole blood passage time {(heparin tube) − (EDTA-2Na + heparin)} and serum levels of myeloperoxidase and adhesive leukocyte number, respectively, even in blood from patients with DM or ACS, who suffered from inflammation. In conclusion we have developed a clinically feasible method for assessing leukocyte rheological activation in whole blood in ex vivo.This study reports a novel method for assessment of leukocyte rheological activation with a new designed microchannel array chip to mimic the human microvascular network for microchannel array flow analysis (MCFAN). Study subjects were 79 healthy volunteers and 42 patients with type 2 diabetes mellitus (DM) and 36 patients with acute coronary syndrome (ACS). Using the anticoagulants heparin and ethylene-diamine-tetraacetic acid (EDTA)-2Na which inhibits platelets and leukocytes by chelating CaThe online version of this article (10.1007/s00380-019-01486-y) contains supplementary material, which is available to authorized users. Abnormal activation states of leukocytes and leukocyte–platelet interactions play key roles in organ injury induced by atherosclerotic disease , 2, diabCohort studies have shown blood viscosity to be a strong predictor of cardiovascular events , particuTherefore, we designed and manufactured a new microchannel array chip, DKAMCM1-60-7-4.5D, to mimic the human microvessel network. Stepwise increases in shear stress may prevent artefactual activation of platelets. The silicon dioxide film on the silicon wafers provides phase contrast that permits clear microscopic observation of blood cells, especially transparent leukocytes and platelets, from passing through the microchannel array without dying .2+ and Mg2+) essential to the activation of platelets and some leukocytes [2O and 60 cmH2O after calibration, according to the duration of saline passage through the channels.Although the number of adhesive leukocytes per unit area has been reported as an index of leukocyte activation –7, its uukocytes , 19. TheThe clinical characteristics of the study participants including 79 healthy volunteers, 42 patients with type 2 diabetes mellitus (DM) and 36 patients with acute coronary syndrome (ACS) are shown in Table We used a plot-type microchannel flow analyser system  equipped with a new microchannel array chip  designed as an ex vivo microvascular model to assess whole blood rheology and leukocyte activity. The detailed procedures and apparatus of BWA-MCFAN have been described elsewhere , 15. In 2O  or − 60 cmH2O . The time required for saline to pass through the microchannels was determined before each blood measurement for calibration. Microscopic motion images of blood passing through microchannels were monitored and stored via computer. When 0.08–0.10 mL of blood had exited the microchannel array, five fields were recorded, five still images were randomly selected for offline analysis, and the numbers of adhesive or clumped leukocytes on the microchannel platform in these images were counted [Within 20 min of collecting blood into the two tubes, 0.1 mL of blood was drawn through the chip under a constant vacuum of − 30 cmH counted  by middl counted . The whoThe study protocol was approved by the Ethical Committee of Dokkyo Medical University Nikko Medical Center. All procedures performed in this study were conducted with informed consent of the patients and complied with the national ethical guidelines for medical and health research involving human subjects and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.–9 or 10–10 M) was added into whole blood from healthy subjects (n = 10) in the heparinized tube and EDTA-2Na + heparin tube. After 5 min incubation with FMLP, the whole blood passage time was measured by BWA-MCFAN.All participants arrived in the laboratory at 2:00 pm. The participants had abstained from alcohol, caffeine, and smoking for at least 12 h prior to the study. All participants drank 200 mL of water and remained seated at rest for 5 min before blood sampling. Blood (10 mL) was sampled from the antecubital vein, and 5 mL was collected into each of two tubes: a 5% vol heparin tube  and an EDTA-2Na + 5% vol heparin tube . FMLP  was used to induce leukocyte activation experimentally , 20. FMLt test or 2-way analysis of variance. Correlations were assessed using Fisher’s coefficient (r). A p value < 0.05 was considered statistically significant.Data were statistically analysed using JMP 14.0 J software . Continuous variables are described as the mean ± standard deviation (SD). Differences were analysed with Student's 2 each using a dicing saw  to obtain the microchannel chips. The resulting pattern was carefully examined with a scanning electron microscope  -2Na, are demonstrated in whole blood ex vivo Fig. . A bluepan) Fig. k and conan) Fig. l. SchemaN-formyl-methionyl-leucyl-phenylalanine (FMLP) ex vivo. FMLP was added to fresh whole blood from healthy subjects to activate leukocytes experimentally [First, we determined whether our new BWA-MCFAN-equipped method with DKAMCM1-60-7-4.5D could quantify leukocyte activation induced by mentally , 20, andn = 79, Table 2O and − 60 cmH2O . There were variations in the number of adhesive leukocytes per field in heparinized blood below − 30 cmH2O . Numbers of adhesive leukocytes in blood with heparin + EDTA-2Na were remarkably lower than those in blood with only heparin under both suction pressures  in healthy subjects (n = 79) than in patients with Type 2 diabetes mellitus  and acute coronary syndrome   . This artefactual activation of platelets downstream of capillaries induced by conventional microarray chips with abrupt narrowing may prolong to the whole blood passage time somewhat .2+, leukocytes can transform to an active state with pseudopods and increased viscoelastic coefficients [2+ levels in human neutrophils in a concentration-dependent manner, as determined by Fura-2 imaging and reactive oxygen intermediates production [2+ significantly increased the adhesive leukocyte number, and EDTA-2Na inhibited FMLP-induced activation of leukocytes and platelets by scavenging Ca2+. EDTA inhibits leukocyte activation by (1) inhibition of Ca2+-dependent activation of leukocytes, and (2) inhibition of platelet–leukocyte interaction. The inhibition of activation of leukocytes and platelets by EDTA-2Na was associated with an increase in leukocyte deformability and a decrease in leukocyte adhesion to the chip and platelets. The change in the passage time of whole blood, which is significantly correlated with the adhesive leukocyte number and plasma levels of MPO, was calculated using BWA-MCFAN by subtraction of the passage time of whole blood treated with EDTA-2Na + heparin from that of blood treated with heparin only, even in blood from patients with DM and ACS, who suffered from inflammation. This technique may provide a clinically feasible method for determination of leukocyte rheology. The rheology of leukocytes has significant implications for their functional behaviour, including flow through, microcirculation, and interaction with endothelial cells [2O and/or 35 cmH2O.In the presence of Caficients , 23, 24.oduction . In the al cells . The optIn conclusion, we have developed a clinically feasible method for assessing leukocyte rheology in whole blood using BWA-MCFAN with a new silicon chip designed to mimic the human microvascular network. Using the anticoagulants heparin and EDTA-2Na, we were able to quantify leukocyte rheology and show that the change in whole blood passage time, as calculated by subtraction of the passage time of blood treated with both heparin and EDTA-2Na from that of blood treated with heparin only, is correlated with adhesive leukocyte number.2O. Note that many activated leukocytes adhered on the terrace and clumped in the capillaries. As a result, whole blood passage time was prolonged (57 s). Videos 1 and 2 feature samples from the same subject with a body mass index of 22, haematocrit of 47, and white cell count of 7330/ml (MP4 1387 kb)Supplementary file1 Microscopic video demonstrating heparinized whole blood, from a 30-year-old healthy male current smoker, passing through the microchannel array under − 30 cmH2O. Note that fewer leukocytes adhered on the terrace and clumped in the capillaries than in the heparin-only sample featured in Video 1. Whole blood passage time was 43.6 s (MP4 1420 kb)Supplementary file2 Microscopic video demonstrating ethylene-diamine-tetraacetic acid (EDTA)-2Na and heparinized whole blood from the same subject as for the sample in Video 1 passing through the microchannel array under − 30 cmHBelow is the link to the electronic supplementary material.


Our results showed that forest floor had higher C contents and lower N contents in PF compared to NF, resulting in high forest floor C/N ratios and a decrease in the quality of organic materials in forest floor under plantations. In general, soil C, N contents and their isotope changed significantly in the forest floor and mineral soil after land use change (LUC). Soil δ13C was significantly enriched in forest floor after LUC while both δ13C and δ15N values were enriched in mineral soils. Linear and non-linear regressions were observed for MAP and MAT in soil C/N ratios and soil δ13C, in their changes with NF conversion to PF while soil δ15N values were positively correlated with MAT. Our findings implied that LUC alters soil C turnover and contents and MAP drive soil δ13C dynamic.Soil C and N turnover rates and contents are strongly influenced by climates  as well as human activities. However, the effects of converting natural forests to intensively human-managed plantations on soil carbon (C), nitrogen (N) dynamics across various climatic zones are not well known. In this study, we evaluated C, N pool and natural abundances of  Approximately 1,500 ± 230 Gt of soil organic carbon (SOC) is stored in the first meter of soil, nearly twice as much as atmospheric carbon (828 Gt as CO2)2 and almost 44% is stored in forests3. A better understanding of soil C and N balance in forest floor and mineral soil can help guide the implementation of mitigation policies to reduce the emission of greenhouse gases by sources or enhance their removal from the atmosphere by “sinks” which refers to forests, vegetation or soils that can reabsorb CO24. In contrast to the clear inventory-based assessments of aboveground C on both regional5 and global scales3, the C and N pools in forest floor and mineral soils remain uncertain because of the higher variations among global forest ecosystems7.Globally, soil is the largest carbon (C) pool in terrestrial ecosystems9, consequently resulting in the changes of soil C and N cycles12. The impacts of converting natural forest (NF) to plantation forest (PF) on soil C and N cycles have been well examined at the stand scales, relating their variances in above- and below-ground C input through litter-fall, root exudation, and their inherently different management practices15. However, the influence of the wider regional or global scale conversion of NF to PF on changes of soil C and N cycles is not yet well understood. This is perceived as a key bottleneck in improving the prediction and feedback of soil C and N mitigation related to land use change (LUC). Previous studies have suggested that the impact of converting natural forests to plantations significantly impacts the quantity and quality of C and N input between natural forests and plantations15. Finzi et al.10 for example, reported that changes in the species composition control forest floor and surface soil C and N dynamics while converting natural evergreen broadleaf forests to moso bamboo plantations significantly decreased the concentrations of C within the 0–40-cm.Natural environmental factors and human activities generate abrupt, large scale, irreversible changes and thus alters forest composition13C and 15N (expressed as °/oo) has uniquely been used to estimate soil C turnover rate20. The 13C and 15N abundance in soils is a dynamic function of the rate and isotopic composition of inputs and outputs, and the internal C, N transformations that occur in a soil system. It can also be used as a gross indicator of environmental processes that impact soil C storage in forest ecosystems and therefore provide integrated insights into soil C and N cycles22. Litter inputs generally lowers soil δ13C and δ15N values while higher decomposition leads to increased δ13C and δ15N values23. Moreover, it has been well evaluated that for sites with stable vegetation and low human disturbance, SOC turnover rates do not significantly change according to vertical soil-profile SOC contents and δ13C24. The values of soil δ15N vary with some soil N cycle processes, e.g., N deposition, soil N nitrification and denitrification, and are used as a tool to examine soil N availability26. In addition, N cycle processes in stand-scale forests altered by human disturbance and climatic factors28. However, details pertaining changing soil δ13C and δ15N with NF converted to intensively managed plantations along climatic gradients of MAT and MAP at a regional scale is still not well understood.The natural abundance of soil 2 encompasses a climatic gradient, from cold temperate to tropical climate zones, and therefore has diverse land uses (LU) ranging from deciduous forests in the north to evergreen forests in the south29. China can be viewed as a unique “laboratory”, with its complex interactions between the varied climatic zones and intensive human activities, thus providing an excellent opportunity to examine simultaneous climate and human impacts on the forest soil C and N pools30. To date, several studies have been carried out to evaluate soil C pools across China’s forests using statistical or biogeochemical models32. However, there are some inconsistencies in soil C and N pool estimations such as the increasing of plant cover through reforestation and afforestation programs, the lack of data from repeated inventories, the variety of methods used to assess the carbon balance of China and the variation of climate conditions within those studies which vary from one period to another. Among others, these are either because of the insufficient observations or inconsistent measurement methods among the studies32. Recently, Tang et al.33 conducted a field survey involving 14,371 field plots to evaluate the current C stocks in mineral soil of China’s terrestrial ecosystems in order to examine their biogeographical patterns and potential climatic drivers. However, the aforementioned studies evaluated soil C pools only in the NF. The increasing needs for timber and other economic forest products in China, means that large areas of NF has been converted to intensively managed plantations35 but there is still significant uncertainty concerning changes in forest floor and mineral soil C and N stocks and turnover processes potentially induced by forest conversion along the 4200 km transect from northern China to southern China.China, with a land area of 9.63 million km−1) and δ13C and δ15N values for 1 meter vertical depth in NF and PF in forest floor and mineral soil, (ii) quantify changes in soil C and N contents and turnover rates as induced by LUC, and (iii) determine the effect of climate factors on the patterns of soil C and N contents, and natural abundance of soil 13C and 15N after LUC. We hypothesize that: (i) C, N, δ13C and δ15N contents in forest floor would significantly differ between NF and PF, and vary significantly among the study sites in the climate gradient, (ii) site specific content and turnover times of soil C and N would be increased by LUC, but (iii) overall climate controlled variations in soil C, N, δ13C and δ15N content between site would not be significantly affected.The objectives of this study are, in a range of climate zones in eastern China to: (i) determine the patterns of soil C and N contents  and their values were considerably affected following conversion of natural forest (NF) to plantation forest (PF)  in soil C, N content and C/N ratio among sites (Table P < 0.05) altered following the conversion from NF to PF but soil N did not change significantly (P = 0.214). In addition, we observed a gain of the mean values of soil C content over 1 m by +6% (HT), +39% (XY) and a loss varied between −38% to −12% at DH > MH > JF > QY  alters C, N values and C/N ratio. However, the conversion from NF to PF strongly influenced C and C/N ratio   differences in forest floor δ13C and δ15N values among sites. In addition, LUC significantly increased forest floor δ13C values (Table δ15N (P = 0.155).Forest floor δ13C and δ15N values significantly (P < 0.001) varied among sites in mineral soil after the conversion from NF to PF. In addition, soil depth, LU and site × soil depth interaction were significantly altered after the conversion from NF to PF  ranged from −4.14 °C at MH to 21.08 °C at DH, and mean annual precipitation (MAP) increased from 436 mm to 2499 mm from northern to southern China Table . We obse PF Fig. , with a  PF Fig. .Table 5Lδ13C and δ15N in forest floor layer and mineral soil layers over 1-meter depth under NF and converted plantations across eastern China. Firstly, we found high forest floor N, but low C and C/N ratios in forest floor layer under NF as compared to PF. This point to the better quality of leaf litter materials in NF stands and large release potential of N during decomposition of the litter. This result was consistent with that found by Chen et al.35 who suggested the lower C/N ratios in forest floor litter in NF compared to hoop pine (Araucaria cunninghamii) plantations in Australia. The increase in forest floor C and C/N values and decrease in forest floor N may indicate that forest management practices provide favorable conditions for decomposing microorganisms thus alter their values as previously reported. In China, forest management practices have been directed towards timber production and sustained yield of wood supply, a practice which has significantly altered the balance between heterotrophic litter decomposition and litter inputs, thus impacting on the C content in the forest floor. This trend is consistent with the results of Vesterdal et al.36 who reported that the accumulation of nutrients in the forest floor is altered with increasing thinning intensity. There were significant differences in C, N and C/N within the study sites because of the effects of the forest conversion, including tree species that may affect the decomposition rate and the turnover of these elements in the soil38. Tree species differ in their carbon sequestration potential, LUC by changing species composition, tree density and forest structure altering their sequestration potential. Similar results have been reported by Vesterdal et al.39 who observed that forest floor C and N contents and C/N ratio were strongly affected by trees species. Besides, Fonseca et al.37 reported that forest floor C and N under the coniferous species had a large quantity of organic materials poorly decomposed, while a high rate of transformation of forest floor and incorporation in mineral soil have been observed in broad-leaved species. In our study, the forest floor litter was mainly composed of deciduous or evergreen broad-leaf litter in NF, while forest floor in PF is almost purely needle litter. Broad leaves are generally thought to produce mull forest floors that are richer in nutrients and promote rapid decomposition40. In addition, the higher forest floor N observed in NF at MH and QY is probably a result of the low temperature in North part of China which negatively affects the decomposition process and consequently leads a larger accumulation of organic matter. Thus, this study revealed that forest floor C and N content is strongly affected by human disturbances, consequently by LUC.The present study provides a quantitative overview of C and N contents, and natural abundances of 41 who reported that the variation of soil C with depth in the profile varies strongly with vegetation type. Ramesh et al.42 also noted that quality and quantity of different soil organic carbon pools change with time depending on the rate of photosynthetic C addition and their losses through decomposition. Our study therefore suggested that LUC alters the carbon-holding capacity of soil in short carbon retention capacity of soil. Furthermore, the changes of soil C and N contents after NF converted to PF were not obvious. We observed a gain in soil C values at XY and HT sites and loss at DH, MH, JF, QY sites after the conversion from NF to PF suggesting that LUC from NF to PF influences C inputs. Similarly, Lewis et al.43 found that the effects of change from NF converted to introduced Pinus sp. plantations were highly site-specific and might have a positive, negative, or no influence on the variation of soil C values. Smith et al.34 also demonstrated that conversion from natural Amazonian forest to plantations altered soil organic C with an increase in surface under Euxylophora paraensis Hub. plantation and decreases under Pinus caribaea var. honduensis Barrett and Golfari. The result could be related to different quantity and quality of C input through root exudation, litter inputs and different management practices between NF and PF44. Although biological N fixation is the primary source of nitrogen input45, soil N values did not change significantly within the study sites after the conversion from NF to PF in the present study indicating that LUC did not alter significantly the balance between N input and loss.In mineral soil, C values were significantly altered after the conversion from NF to PF among site probably because of the difference of soil type, vegetation and trees species. The vegetation cover influences the storage of its elements in that it reduces the arrival of solar radiation directly to the soil. Yet, litter decomposition rates are controlled by the temperature and moisture which directly affect soil microbial activity. These findings are consistent with those of Jobbágy & Jacksonet al.46 who reported 50% the amount of OC located in the upper 30 cm of the soil organic carbon in the layer 0–100 cm. This result suggests a greater soil C sequestration within 0 to 20 cm soil depth. Furthermore, we found that soil C and N content decreased with soil depth may be due to the vertical distribution of roots. Indeed, Jobbágy et al.41, found that root distributions affect the vertical placement of C in the soil, and above- and below-ground allocation affects the relative amount of C that eventually falls to the soil surface from shoots. In addition, it well known that N status is a crucial factor driving forest soil C dynamics and high N availability can promote a greater soil C sequestration47. This trend is in line with our study which reported a significant and positive correlation between C and N probably through the effects on organic matter decomposition45.In general, soil C and N where stored in the 0–20 cm depth segment of the overall profile, i.e. 67 and 57%, respectively. This is consistent with the results found by Batjes δ13C and δ15N have been used as powerful index to evaluate the long-term alterations of C and N cycles22. In the present study forest floor δ13C were enriched after LUC in subtropical and tropical regions while δ15N depleted. The depleted natural abundance of 13C and 15N in litterfall could account for low values of forest floor δ13C and δ15N in NF because of the generally higher above-ground litterfall input in NF compared to PF48. Moreover, we found that soil δ13C were strongly enriched at XY and HT sites after LUC. These findings might also be related to the less above-ground litterfall and below-ground roots inputs in these sites. Hertel et al.49 confirmed this trend by reporting that the conversion of tropical forest into plantations decrease C flux with fine root mortality to soil organic C pool. Moreover, soil organic C and N accumulation and stability have been suggested to be strongly influenced by litter quality, resulting in more stable organic C accumulated in NF soils with high quality of litter substrates50. On the other hand, the intensive management practices in PF  accompanied by higher temperature and precipitation conditions in tropical and subtropical (at the DH and JF sites) regions could enrich soil 13C and 15N in plantation because of more soil C and N loss44. However, soil organic C accumulation and stability were also influenced by soil matrix structure51, and were related to the saturation of SOC, climatic zones and ecosystem types52. It was difficult to explain clearly the effect of LUC on soil δ15N variation within the study sites because of the complex interactions between abiotic and biotic factors.Stable isotopic abundances of δ13C and δ15N with soil depth53. In our study, we observed an enrichment of soil δ13C following the increase of depth layer by 3.09‰ in surface mineral soil (0–10 cm) after LUC probably because deeper soil layer has a greater humification of organic matter decomposition or largely to the increasing residence time of the organic C in the soil54. This result was in line with Brunn et al.20 who found the mean 13C enrichment between δ13C values of the Oi horizon and δ13C values of soil organic matter (SOM) in 10 cm soil depth was 3.4 + 0.2‰ under temperate beech forests. Several processes might cause enrichment of δ13C with increasing soil depth e.g., the depletion of 13C in the atmosphere due to combustion of fossil fuel, and the considerable presence of litter and roots with depleted 13C in the upper soil. Accoe et al.55 observed that the average rate of change in soil δ13C is directly related to organic matter decomposition rates in different parts of the soil profile. Thus, the extent of change in 13C-abundance with increasing soil depth may indicate the quality or stability of SOM under continuous C3 vegetation55. Furthermore, isotopic fractionation during microbial metabolism of SOM and the downward cycling of hydrophilic 13C enriched decomposing products with dissolved organic carbon fluxes possibly contributing to the establishment of the vertical δ13C depth trends56. The pattern of soil δ13C observed in PF of DH showed an increase from 0 to 40 cm and then a decrease until 100 cm depth layer reflecting the more complex processes of microbial degradation and mixing of soil C of different ages57. On the other hand, the low soil δ15N values observed at the surface soil across the study sites particularly at XY could be due to the continuous addition of plant residues with extremely low δ15N values58.A number of studies observed an increasing tendency of 60, and natural abundance of soil 13C22 and 15N62. There were linear and non-linear regressions of soil C/N ratios and δ13C with MAT and MAP in both NF and plantations, which indicated that SOC turnover rates were largely determined by MAT and MAP. Our result was consistent with Wang et al.22 who examined large-scale controls of climate over patterns of SOC turnover across terrestrial biomes worldwide using a meta-analysis of soil δ13C in previous literatures and demonstrated that SOC turnover was substantially accelerated with increasing MAT. In addition, the distribution of soil δ13C was slightly correlated with MAP after LUC probably due to the replacement of vegetation types which determines changes in the relative vertical distribution of soil C along rainfall gradients41 after LUC. This result is in line with those found by Burke et al.63 who reported that precipitation clearly has a direct role regionally and globally in the amount of soil C stored. Thus, our findings revealed that MAP is a key factor controlling soil C accumulation and decomposition after the conversion from NF to PF. According to Jia et al.64, vegetation type and soil type rather than MAT explained the variability in soil δ13C along the 400 mm isohyet in China.Climatic variables have strong impacts on soil organic C and N contentsδ15N61. Contrary to these results, we did not observe clear relationship between climate factors and δ15N in the present study. This could be due to the climate change which may change soil δ15N values through altered precipitation patterns, elevated temperatures, and more frequent and extreme weather events. In addition, climate change can affect temperature-sensitive biogeochemical processes, including N mineralization, nitrification and denitrification, soil respiration, litter decomposition, as well as root dynamics and plant productivity65, consequently, altering the rate of δ15N accumulation.Previous studies suggested that climate is the primary factor controlling soil In August 2017, soil samples were collected from one natural forest (NF) and one plantation forest (PF) stand at six sites including Mohe (MH), Qingyuan (QY), Xinyang (XY), Huitong (HT), Dinghushan (DH), Jianfengling (JF) across eastern China forest Fig. , Table 5δ13C and δ15N.Forest floor litter samples were oven-dried at 65 °C for 72 h, and mineral soil samples were air-dried at room temperature for 2 weeks before passing through a 2 mm sieve to remove coarse stones, plant residues, and roots. Then, the forest floor and soil samples were ground to fine powders using a ball mill  for measurements of soil C and N contents, and 13C and 15N composition were measured using an isotope ratio mass spectrometer (IRMS) , connected to a CN elemental analyzer . Carbon and nitrogen stable isotope abundances were calculated as δ13C and δ15N (‰) using the following formula:Soil C and N pools and stable isotope sample is the13C:12C or 15N:14N ratio in the samples and Rstandard is the 13C:12C or 15N:14N ratio in the standard. The Vienna Pee Dee Belemnite (VPDB) and atmospheric N2 (δ15N = 0.0‰) were used as the standard, respectively. The precision of isotopic composition was checked using internal standards i.e. acetanilide, L-histidine, D-glutamic and glycine28. In general, the analytical precision for δ13C and δ15N was better than 0.2‰.where RP < 0.05 unless otherwise stated. The sample differences of δ15N, δ13C values, C and N concentrations, the effect of LU and depth on C, N, δ13C, and δ15N in whole soil and the comparison of C and N among LU types and depths were tested with analysis of multivariance (ANOVA). Linear regression and nonlinear regression analysis were used to test the relationships of C/N, soil δ13C with climatic factors  across forest type. The samples from each depth were used as dependent measurements in the regression. All statistical analyses were performed with the SPSS version 20.0 66.The results are the average of the replicates determined from three subsamples of the same site. Statistically significant differences were determined by


Current approaches aimed at inducing immunogenic cell death (ICD) to incite an immune response against cancer neoantigens are based on the use of chemotherapeutics and other agents. Results are hampered by issues of efficacy, combinatorial approaches, dosing and toxicity. Here, we adopted a strategy based on the use of an immunomolecule that overcomes pharmachemical limitations.Cytofluorometry, electron microscopy, RT-PCR, western blotting, apotome immunofluorescence, MLR and xenografts.We report that an ICD process can be activated without the use of pharmacological compounds. We show that in Kras-mut/TP53-mut colorectal cancer cells the 15 kDa βGBP cytokine, a T cell effector with onco-suppressor properties and a potential role in cancer immunosurveillance, induces key canonical events required for ICD induction. We document ER stress, autophagy that extends from cancer cells to the corresponding xenograft tumours, CRT cell surface shifting, ATP release and evidence of dendritic cell activation, a process required for priming cytotoxic T cells into a specific anticancer immunogenic response.Our findings provide experimental evidence for a rationale to explore a strategy based on the use of an immunomolecule that as a single agent couples oncosuppression with the activation of procedures necessary for the induction of long term response to cancer. To this end, much study during the past decade has been directed towards the search for agents that by inducing immunogenic cell death (ICD), an apoptotic program which includes procedures that incite an immunogenic response against cancer neoantigens, would consequently establish a state of tumour specific immunosurveillance.4 but, despite results of interest, only a few of the compounds have been found to fulfil all canonical requirements for ICD induction and fewer still to have the ability to be both therapeutical and to induce ICD, hence calling for combinatorial approaches which have reflection on toxicity, dosing and therapeutical scheduling.4Anti-cancer drugs, toxic agents and a variety of other agents have been experimented5Here we hypothesised that if an ICD process is an integral part of a natural cancer surveillance program, the candidate element(s) that induce ICD may be transposed to therapeutic use without the uncertainties and the collaterals of pharmacological agents. To test this hypothesis, we have adopted a strategy centred on the use of a molecular component of the immune network with a candidate role in cancer immunosurveillance.6 which has cytostatic properties and selective anti-tumour properties.11 While arrested normal cells preserve the ability to resume proliferation after βGBP treatment,7 arrested cancer cells regardless of mutational load undergo apoptotic death.11 βGBP operates through mechanisms that involve high affinity receptor binding (Kd ~ 1.5 × 10−10 mol/L7) and molecular interactions leading to functional inhibition of the p110 class1A and class 1B PI3K catalytic subunits.12 Consequent downregulation of PI3K activity has two major outcomes which are reversible in normal cells but not in cancer cells: suppression of Ras-GTP loading leading to block of ERK activation12 and negation of akt gene expression leading to loss of Akt10 function, conditions that either by blocking the ability of cancer cells to proliferate or by impairing their ability to survive can block oncogenicity. These effects highlight two fundamental properties: a direct and selective anticancer action transferable into therapy and a physiological participation in cancer surveillance. Therapeutically human βGBP has been proven to strongly reduce human Kras-mut/TP53-mut colorectal cancer xenograft growth as a single agent11 and, as a single agent, to drive to apoptotic death a variety of cancer cells from solid tumours, including cells sourced from colon, pancreas, prostate and breast which bear Kras mutations and tumour suppressor deficiency.5We have utilised the15kD β-galactoside-binding protein (βGBP) a molecule primarily produced by activated CD8+ T cells, by CD8+ memory cells and by activated CD4+ T cells,15 calreticulin (CRT) transfer from the lumen of the ER to the surface of the cancer cell and the release of ATP by the dying cancer cell19 and, consequent to these events, we have assessed dendritic cell (DC) activation, and found that the stated requirements for ICD induction were met.Here we have investigated key canonical events which are fundamental to ICD induction. We have focused on endoplasmic reticulum (ER) stress, autophagy,Our data provide a rationale for exploring a new strategy based on the use of a physiological component of the immune network that as a single agent couples oncosuppression with the activation of procedures that lead to ICD induction.11 Human recombinant βGBP was expressed in Escherichia coli BL21 (DE3) using hGal-1 cDNA in PET21a, purified by lactose-agarose (Sigma) affinity chromatography and purity assessed by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF).SW620 and SW480 human colorectal cancer cells from the American Type Culture Collection were authenticated and cultured as detailed previously.6 cells were implanted s.c. and grown to a tumour size of approximately 40 mm3. Mice were injected s.c. in the tumour area with 150 μl of βGBP from a 5 μM stock solution, or PBS in controls, six times each week and sacrificed after 5 weeks. Experimental details, ethical guidelines and authority approval have been reported previously.11SW620 xenografts were grown in thymectomised CD-1 female nude mice (Charles Rivers Laboratory). 5x10Samples were fixed in 2% glutaraldehyde in PBS for 24 h at 4 °C, post fixed in 1% osmium tetroxide for 2 h and stained for 1 h in 1% uranyl acetate. Samples dehydrated in acetone were then embedded in Epon-812. Ultrathin sections (60 nm) were cut with a Reichert ultramicrotome, counterstained with uranyl acetate and lead citrate, and examined with a Philips CM10 transmission electron microscope.20RNA extraction, reverse transcription method, specific primers and conditions for PCR amplification of CHOP, BiP, XBP-1 and HPRT have been reported in previous work.Cells were lysed, and protein concentrations were assessed according to standard procedures. Anti-p62/sequestosome1 antibody (BD Transduction Laboratories) followed by horseradish peroxidase-conjugated goat anti-mouse antibodies (Santa Cruz) was used to evidence the p62/sequestrosome1 protein.Cells were fixed in 4% paraformaldehyde and to visualise LC3, an anti-LC3 polyclonal antibody (Abgent) followed by FITC-conjugated goat anti-rabbit antibodies (Life Technologies) was used. Calreticulin polyclonal antibodies (Affinity Bioreagents) and Texas Red-conjugated goat anti-rabbit polyclonal antibodies (Jackson Immunoresearch Laboratories) were used to visualise calreticulin. FITC-labelled wheat germ agglutinin was used for cell surface staining and CD1a monoclonal antibody (BD Pharmingen) followed by FITC-conjugated goat anti-mouse antibody (Life Technologies) was used to visualise the CD1a glycoprotein. In live cells monodansylcadaverine (Molecular Probes) was used as an autofluorescent vital dye. DAPI (Sigma Aldrich) was used to visualise nuclei. At least three independent experiments were carried out according to standard and manufacturer’s recommended procedures and analysed using ApoTome Axio Observer Z1 inverted microscope (Zeiss) equipped with an AxioCam MRm Rev.3. Co-localisations were assessed with Axio Vision software, release 4.6.3 (Zeiss).Cells were cultured with or without βGBP for 48 h in the presence of ATPase inhibitor ARL 67156 (Sigma Aldrich), centrifuged at 1500 rpm for 5 min, supernatants recovered, and extracellular ATP levels measured by the luciferin-based ENLITEN ATP assay (Promega) according to manufacturer’s instructions.6 cells/3 mL in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin G, 100 mg/mL streptomycin, 50 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/mL interleukin-4 (IL-4) (Miltenyi Biotec). Cytokines were replenished every other day, along with 20% fresh medium. SW480 cells grown on coverslips treated or mock treated with βGBP for 48 h were washed three times in PBS to remove βGBP, co-incubated with DC’s for 4 h at 4 °C (1/3 ratio) and finally washed in PBS to remove unbound DCs. The cells were fixed with 4% paraformaldehyde in PBS for 30 min at 25 °C, stained for CRT (Affinity Bioreagents) or the CD1a DC marker (Miltenyi Biotec) and observed as above.To generate monocyte-derived DCs, human peripheral blood mononuclear cells (PBMC), obtained under informed consent from healthy donors, were isolated by Fycoll-Paque gradient (Pharmacia). CD14 + monocytes were positively selected using anti-CD14 antibody-conjugated magnetic microbeads (Miltenyi Biotec). To generate immature DCs, purified monocytes were then cultured in 12-well plates for 6 days, at a density of 1011 Expression of CRT was monitored using mouse monoclonal antibody (Santa Cruz) and phycoerythrin-conjugated anti-mouse antibodies (Becton Dickinson) for 30 min at 4 °C followed by twice washing in PBS. DCs were stained with FITC-conjugated anti-CD86 and anti-CD83 antibodies (Becton Dickinson) for 30 min at 4 °C followed by two washes in PBS. Cells were gated according to FSC and SSC parameters. Appropriate isotype-matched control antibodies were included in the assessments and propidium iodide staining was used to evidence dead cells. At least 5,000 viable cells were acquired in each experiment. Acquisitions were performed on an EPICS XL flow cytometer (Coulter).Analysis of apoptosis  has been reported previously.3H-thymidine (1 μC/ml in a 96 well plate) for the last 16 h. In other experiments immature DCs treated or mock treated with βGBP (2 nM) for 24 h, were cultured with allogeneic PBMC for 5 days before adding 3H-thymidine (1 μCi/ml in a 96 well plate) for the last 16 h.MLR was performed on immature DCs incubated for 24 h with βGBP treated or mock treated SW480 cells extensively washed and irradiated (3000 rad) and then cultured with allogeneic PBMCs for 5 days before adding 11 In our experiments we have used the lowest therapeutically effective dose of human recombinant βGBP (2 nM) that induces growth arrest and apoptosis,11 a dose about fifty-fold lower than that required (~100 nM) for βGBP to act as a down-regulatory cytokine during the silencing phase of a T cell immune response.6For our investigations we selected SW620 cells derived from a human metastatic colorectal cancer and SW480 cells from the primary isogenic parent tumour, both carrying a Kras-G12V mutation and biallelic mutations in TP53 (R273H and P309S) and both unresponsive to current therapeutic attacks but responsive to βGBP treatment that arrests their proliferation and forces them into programmed cell death.First, we looked for growth arrest, time related expression of apoptotic parameters and cellular death, along with evidence of ER stress and autophagy, obligatory determinant factors for ICD induction. Figure 23 Using (q)RT-PCR, we monitored the expression of CHOP  and BiP (Grp78), major UPR functional indicators24 and splicing of XBP-1 mRNA, for which a splice variant specific activation of the UPR has been demonstrated.25 As shown in Fig. Next, we investigated whether βGBP treatment would affect the unfolded protein response (UPR), a prominent part of ER stress-induced events which activates autophagy.27 Compared to controls, a conspicuous increase in the number and size of LC3 dots was observed in the βGBP-treated cells , an autofluorescent vital dye that selectively accumulates in autophagic vacuoles. Within the first 48 h of treatment we found evidence of MDC in the cytoplasmic vacuoles of βGBP-treated cells Fig. , arrows.lls Fig. . These rlls Fig.  which sp11 we looked for evidence of autophagic structures in histological sections of SW620 xenografts. In Fig. Having detected evidence for ER stress and autophagy, based on previous evidence showing that βGBP has strong therapeutic efficacy against SW620 xenograft development, to investigate whether the autophagic effect that we had observed in cultured cells could be detected in the in vivo model where tumour mass development is strongly inhibited by βGBP,29 and the release of ATP,18 a process induced by autophagy,27 are necessary events. We, therefore, investigated whether βGBP treatment had affected the expression of CRT and ATP. Using immunofluorescent detection, we found early evidence of CRT exposure at the cell surface. Figure Activation of ICD requires changes in the composition of the cancer cell surface and the release of soluble mediators, processes where shifting of CRT from the ER to the cell surface, a process induced by ER stress,Next, we looked for evidence of ATP release and found that by hour 48 there was clear evidence that ATP had been released by both the SW620 (about a fivefold increase) and the SW480 treated cells (about an eightfold increase) Fig. . Thus, t18 we looked for evidence of dendritic cell activation by investigating whether dendritic cells would interact with cells that had been treated with the βGBP molecule. For this purpose, the tumour cells were stained with antibodies to CRT (red) and the DCs stained with a monoclonal antibody to the CD1a activation marker (green). As shown in Fig. As CRT and ATP operate on dendritic cell receptors to activate dendritic cells (DCs) and support the presentation of tumour antigens to cytotoxic T cellsNext, to determine whether DC activation by βGBP-induced ICD could activate T cells, tumour cells were pre-treated with βGBP for 24 h, washed and co-incubated with DCs in a mixed lymphocyte reaction (MLR). Figure Finally, in addition to the above investigations, to substantiate the premise that, as a physiological molecule, unlike pharmacological inducers which carry associated toxicity, βGBP has no harmful properties, we pre-treated DCs with βGBP for 24 h and carried out a mixed lymphocyte reaction (MLR). Figure 1Therapeutic induction of ICD, a process aimed at inciting the immune system into a response against cancer neoantigens requires the activation of an apoptotic program which through a sequence of events spanning from ER stress to autophagy, to CRT cell surface exposure and ATP release, leads to the activation of dendritic cells.ad hoc therapeutic against aggressive, otherwise therapy resistant cancer cells, is a physiological inducer of procedures that lead to ICD. We show that the apoptotic process induced by the βGBP molecule includes a time-space of growth arrest where ER stress and autophagy take place, where CRT moves to the cell surface and ATP is released, and where DC activation, a necessary step for priming T cells into an anticancer immunogenic response, is occurring. This conclusion is based on the rise of CD86, a specific DC activation marker, on the increase of CD83, a DC maturation marker, on experiments where dendritic cells interacted with cancer cells that had been treated with the βGBP cytokine and on the evidence that DCs co-cultured with βGBP-treated cancer cells positively affected T cell proliferation. Also, we show that unlike pharmacological inducers that carry associated toxicity, βGBP, a physiological ICD mediator, had no harmful properties, as revealed by MLR experiments where βGBP- activated DCs displayed an increased capacity to stimulate T cell proliferation.Here we found that a native element of the immune network which acts as an 4 of the many drugs and agents experimented, only a few have so far been found to fulfil all canonical requirements for ICD induction, and fewer still to have the ability to be both therapeutical and to induce immunogenic cell death as single agents, hence calling for combinatorial approaches which have reflection on toxicity, dosing and therapeutical scheduling.4Current practices for ICD induction are based on the use of cancer chemotherapeutics and a variety of other agents but, although encouraging results at preclinical and clinical level have been reported,4 suggests that chance plays a part both in drug selection and in respect of the drug’s efficacy as it is probably impossible to copy with drugs or other agents the modality of events as they occur when ICD is induced by a T cell effector, as in our study. Unlike drugs and other foreign agents, βGBP, a natural immunomodulator, operates through biological mechanisms and functions that proceed according to a program. They initiate with high affinity βGBP receptor binding followed by PI3K downregulation and signalling hence,12 events that result in cancer cell death through a graded process that allows procedures that lead to ICD to take place.Also, which given compound can be a potential ICD inducer cannot be predicted. The number and the variety of agents experimented so farThe past decade has witnessed a major development in anti-cancer therapies, but strategies that further to killing cancer could also secure long-term protection by instating a state of cancer specific immune surveillance are still missing. Our data provide direct experimental evidence for a rationale to explore the potential of a strategy based on the use of a natural immunomolecule with no innate toxicity that as a single agent acts as a tumour suppressor and an activator of procedures necessary for the induction of long term protection against cancer.


Alkyl fluorides are also directly converted into the corresponding alkyl silanes under similar conditions, even in the absence of the Ni catalyst. Applications of this protocol in late-stage defluorosilylation of potentially bioactive pharmaceuticals and in further derivatizations are also carried out.Direct activation of carbon–fluorine bonds (C–F) to introduce the silyl or boryl groups and generate valuable carbon–silicon (C–Si) or carbon–boron (C–B) bonds is important in the development of synthetically useful reactions, owing to the unique opportunities for further derivatization to achieve more complex molecules. Despite considerable progress of C–F bond activation to construct carbon–carbon (C–C) and carbon–heteroatom (C–X) bond formation, the defluorosilylation via C–F cleavage has been rarely demonstrated. Here, we report an  Transformation of strong C-F bonds into C-Si bonds is an extremely useful strategy for further derivatization of organic molecules. Here, the authors report a nickel-catalyzed strategy to convert aryl fluorides into sylylated arenes while defluorosilylation of alkyl fluorides is achieved under metal-free conditions. Numerous efforts have hence focused on the development of effective fluorination methods, significantly expanding the diversity of available F-containing compounds2. With the steady progress of methods for the preparation of organofluorine compounds, the activation of inert C–F bonds has recently drawn increasing attention, especially with respect to the effective derivatization of organic fluorine compounds5. Compared to the relatively activated C–X bonds of halogenated arenes and their equivalents , which easily undergo oxidative addition in metal-catalyzed coupling reactions, the cleavage of C–F bonds in fluoroarenes (Ar–F) is in general significantly more challenging due to their high bond dissociation energy; they are arguably the strongest bonds that carbon can form , Ar–Cl , Ar–Br , and Ar–I . Thus, not surprisingly, significant research efforts have been directed toward C–F-cleavage protocols in order to develop synthetic strategies for complex aromatic compounds from ubiquitous fluoroarenes.Organic compounds that contain a carbon–fluorine (C–F) bond are attracting increasing attention due to their remarkable applications in pharmaceutical, agrochemical, and organic materials science, which arise predominantly from the intrinsic properties of fluorine (F)10, while methods that employ fluoroarenes have been extended toward the formation of carbon–heteroatom bonds  , as well as relatively high temperatures  Fig. , path b1res Fig. , path b13SiCN), azides (R3SiN3), ally silanes (R3SiCH2CH=CH2), and Ruppert–Prakash reagent (Me3SiCF3), are powerful tools for the direct-transfer functionalization of target molecules. The application of organosilicon compounds in organic electronics, photonics, and biologically active molecules for drug discovery has also been explored owing to their useful physicochemical properties. While a wide range of methods for the synthesis of organosilicon compounds have been developed, methods for the transformation of fluoroarenes into aryl silanes do not exist. Prompted by the versatility and pivotal role of organosilicon compounds16, as well as by our ongoing interest in the activation of inert C–F bonds17, we report herein an available example of the ipso-silylation of aryl and alkyl fluorides via cleavage of unactivated C–F bonds under catalytic conditions mediated by Ni or in the absence of metal catalyst  as the substrate and a Ni catalyst to examine the C–F bond-cleavage reaction with the easily accessible silyl boronate 2a  using Ni(COD)2 , IPr·HCl (20 mol%), and NaOtBu (3 equiv)  product from NaOtBu was not observed either. Encouraged by this highly unexpected result, we decided to focus on this defluorosilylation of fluoroarenes. After screening the reaction conditions  afforded 3a in 89% yield at room temperature , fluorophenyls (1m–1w), fluoropyridines , and polyfluoroarenes (1aa–1cc) were efficiently converted into the corresponding aryl silanes (3a–3cc) using silylborane 2 as the coupling partner. Silylborane 2b furnished 3b in moderate yield under otherwise optimized reaction conditions. The chemoselectivity of this process is nicely illustrated by the fact that functional methoxy , dioxole (3e), silyl ether (3t), phenoxyl (3q), amine (3r), and boronic ester (3v) groups, which could be potentially cleaved in the presence of the Ni(0) complex, remained intact under the applied conditions26. The steric hindrance of ortho-substituents was also tolerated, as shown by the formation of aryl silanes 3j and 3u. It should be noted that fluoroarenes bearing pyrrole, thiophene, or indole rings also participated in the reaction, furnishing the desired defluoroarylsilanes 3f, 3g, 3h, and 3k in good yield, without observation of the expected sp2 C–H bond activation–silylation products27.With the optimal reaction conditions in hand  were successfully prepared using reaction conditions identical to those for π-extended fluoroarenes, despite the different electronic state of the arenes, including high electron-donating NMe2 substitution (3r). These results indicate that a nucleophilic aromatic substitution is highly unlikely to occur in this reaction. The reactivity of simple fluoroarenes was comparable to that of π-extended systems in this ligand-free and mild Ni-catalyzed defluorosilylation, rendering it a highly advantageous protocol for the preparation of aryl silanes. Particularly noteworthy is that fluoropyridine substrates  can be transformed under these conditions. Substrates bearing more than one fluorine group (1aa–1cc) were also successfully monosilylated by carefully adjusting the stoichiometry of the reaction.Notably, a series of substituted aryl silanes , secondary fluorides , and an alkyl fluoride (4l) were smoothly transformed into the corresponding silylboranes using only KOtBu as the activation agent. Defluorosilylation of fluorenyl fluoride 4i and (1-fluorocyclobutyl)benzene (4k) was achieved using Ni(COD)2 as the catalyst to provide alkyl silanes 5i and 5k, respectively. Remarkably, in the case of 4k with tetra-substituted C–F bonds at the cyclobutyl ring, the silylation position was shifted to the neighboring secondary carbon to provide 5k, presumably due to the steric hindrance. In all the cases, C–H bond silylation products were not detected27.Prompted by our findings, we speculated that such a defluorosilylation strategy could also be extended to fluoroalkane substrates (7 was synthesized in moderate yield (41%) under the same reaction conditions. Similarly, pitavastatin analogue 8, bearing an electrophilic carbonyl group and an N-heteroaromatic ring, which in some cases may reduce the efficiency of metal catalysts, was converted into 9 in 50% yield. These results clearly demonstrate the potential utility of this defluorosilylation protocol as a platform for the late-stage functionalization of drugs. The silylated products may potentially serve as useful synthetic building blocks for versatile transformations, and some selected synthetic applications of representative silanes obtained in this work are demonstrated 2 as a catalyst and PhI(OCOCF3) in AcOH -F bond of 4c was successfully achieved in moderate yield by the sequential reaction, i.e., via a Ni(COD)2-catalyzed defluorosilylation, followed by a C(sp3)-Si bond activation to incorporate CO2 under promotion of a fluoride anion  in the presence of an alkoxy base has been previously reporteduct Fig. . As has uct Fig. , borylat20) as the substrate. Under standard catalytic conditions, the reaction of 20 with Et3SiBpin afforded defluorosilylated 21 (51%), while cyclization products were not detected , toluene, and 1-hexene 2 in the presence of a second mole of KOtBu to generate the active catalyst complex D under concomitant release of stable [BPin(OtBu)2]K. The process from 2a to D, i.e., the first step in Fig. 34. We confirmed this reaction process by the detection of intermediate A by 11B NMR studies , assisted by a transfer of the π-bond density of the aryl ring to the d-orbital of nickel 2. 19F NMR experimental studies support the generation of KF (−213 ppm) and show two new peaks (−114 ppm and −135 ppm) for the potential intermediates such as E, F, G, and H during the transformation  disappeared within 20 min. The peak at −114 ppm remained for 60 min and gradually disappeared over 20 h under concomitant emergence of a peak for KF (−213 ppm). Another peak at −135 ppm appeared after 40 min and disappeared after 60 min. Although more studies are necessary to fully interpret these results, transition states/charged intermediates such as η2-arene nickel complexes E, F, G, H, and I may be involved in the activation of the C–F bond.The ability of internal nucleophilic aromatic substitution (path a) is attributed to the A conventional cross-coupling mechanism is also possible, in which C–F bond cleavage proceeds via an oxidative addition of aryl fluorides to Ni(0), followed by a transmetalation and a reductive elimination 2. These reactions afforded only aryl cyclobutene 4k′ via an elimination pathway 2 2 base complex D to generate the intermediate η2-arene nickel complex J39. Subsequently, J is subject to an addition and reductive elimination to afford the corresponding silane (5k).In the case of alkyl fluorides, the reaction does not depend on the presence of the Ni catalyst. The results indicate that the highly nucleophilic, silyl anionic species ess Fig. . Especiaism Fig. . InitialWe have presented an efficient Ni-based catalytic system that is capable of activating the C–F bonds of fluoroarenes in the absence of additional ligands to provide aryl silanes in good to high yield. This protocol for the preparation of aryl silanes is characterized by its mild conditions and wide substrate scope of inert fluoroarenes. The defluorosilylation of alkyl fluorides is directly achieved under very similar conditions, even in the absence of a Ni catalyst. Given the widespread presence of fluorinated compounds in commercially available chemicals, drugs, and functional materials, and the wide range of applications of organosilicon compounds, this strategy should become a powerful tool in organic synthesis, especially for the structural design and potential late-stage functionalization of drug lead compounds.1 , silylborane 2 , Ni(COD)2 , KOtBu  and c-hexane/THF  in a glovebox filled with argon gas. The tube with the mixture was sealed and removed from the glovebox, and stirred at room temperature for 2–12 h. The reaction progress was monitored by TLC. Then, the mixture was added saturated aqueous ammonium chloride (3 ml) and extracted with EtOAc (3 × 3 ml). The combined organic extract was dried over MgSO4 and filtrated. The filtrate was concentrated under reduced pressure. The residue was purified to give the silanes.To a flame-dried screw-capped test tube were sequentially added a fluoroarene 1 , silylborane 2a , KOtBu  and c-hexane/THF  in a glovebox filled with argon gas. The tube with the mixture was sealed and removed from the glovebox, and stirred at room temperature for 2–12 h. The reaction progress was monitored by TLC. The mixture was then added saturated aqueous ammonium chloride (3 ml) and extracted with EtOAc (3 × 3 ml). The combined organic extract was dried over MgSO4 and filtrated. The filtrate was concentrated under reduced pressure. The residue was purified to give the silanes.To a flame-dried screw-capped test tube were sequentially added a fluoroalkanes Supplementary InformationPeer Review File