The chemical composition and structural characterization of the pretreated bamboo fibers were comparatively studied using spectroscopic and wet chemistry methods. Furthermore, the comparative hydrolysis behaviors of pretreated bamboo were studied due to the synergistic interaction between cellulases and xylanase. The NaOH treatment increased the holocellulose contents to 87.4%, and the mean diameter of the cellulose fibers decreased from 50 ± 5 µm (raw fiber bundles) to 5 ± 2 µm. The lignin content and the degree of cellulose polymerization both decreased, while the crystallinity index of cellulose and thermostability increased. The hydrolysis yields of NaOH pretreated bamboo at 135 °C increased from 84.2% to 98.1% after a supplement of 0.5 cellulose to 1 mg protein/g dry xylan. The NaOH pretreatment achieved optimal enzymatic digestibility, particularly at higher temperatures as indicated by the results.In the present study, three pretreatments of sodium hydroxide (NaOH), sulfuric acid (H Bamboo is a perennial herb with rapid growth, short retreading, and easy reproduction. It has been reported that about 30–35 million tons of bamboo are produced per year in Asia. Annual production of bamboo in China reached 1356 billion culms . In receThe enzymatic hydrolysis of lignocelluloses is improved by using an organic solvent, such as glycerin, as a pretreatment and solvent . Romaní Reducing the high input of chemicals and/or energy was essential to develop the energy-saving equipment for the pretreatment and bioconversion of biomass . A sand 2SO4), alkaline (NaOH), and glycerol pretreatments on the structural and hydrolysis behavior of a bamboo biomass at two different temperatures in a sand bath heating medium. The structural analysis of pretreated bamboo was by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), and solid-state cross polarization/magic angle spinning (CP/MAS 13C NMR). The effectiveness of the pretreatment was further verified by the series of structural analyses described above. The synergistic interaction between cellulases and xylanase (XYL) during hydrolysis of the pretreated bamboo samples was also investigated. The enzymatic digestibility of the bamboo after the NaOH pretreatment was explored. The desired enzymatic hydrolysis was achieved in an acetic acid and alkaline pretreatment system with a sand bath device. The results will provide a basis for further improvements in utilizing wood resources and realize the development and utilization of biomass energy by hydrolysis saccharification.The main aim of this study was to study the influence of acidic . The bamboo was sealed in a zip pocket and maintained at 4 °C until use.2SO4, NaOH, NaClO2, toluene, methanol, ethanol, and glacial acetic acid were purchased from Fisher Scientific  and used as received. The pretreatment equipment included an electronic sand bath pot and a thermometer. A bamboo total sample of 100 g oven-dry basis was used for the pretreatment experiment. The commercial enzyme preparations Celluclast 1.5 L, Novozyme 188 , xylanase preparation , and other chemicals used in this study were purchased from Sigma.The chemical reagents, such as H2SO4, 10% NaOH, or 10% glycerol) and placed in a bath at 117 °C and 135 °C for 1 h , which was obtained using the following equation: −1, spectral resolution was 4 cm−1, and the scans were performed 64 times.A NicoletiS10 Fourier transform infrared spectrometer sold by Thermo Fisher  was used under the following test conditions. The spectral range was 4000–400 cmThe crystallinity of the bamboo samples was determined by XRD using the Rigaku Ultima3 x  with scanning parameters of Ni-filtered Cu Kα radiation (λ= 1.54060).This study used the Scherer formula  to calculate d and CrI, as follows:13C NMR measurements were recorded at 100MHz on a Bruker AV-400 spectrometer  with a wide bore magnet and a 4 mm CP/MAS probe. 13C CP/MAS data were collected at 20 °C with 50 kHz spectra width, 2-ms cross-polarization contact time, and 0.035-s acquisition time. The number of scans was 8196, and rotational velocity of the sample was 12 Hz. The data were processed using Bruker Top Spin Software.Solid-state CP/MAS The surface morphology of the bamboo samples was observed using SEM . The test sample was coated with gold using a vacuum sputter coater prior to the SEM analysis.The TG and differential thermogravimetric (DTG) curves of the raw and pretreated bamboo samples were characterized through TG analysis with a TA Q50 Analyzer . Approximately 2 mg of sample was used for the TG/DTG analysis. The samples were heated from room temperature to 600 °C under a flow of 60 mL/min of nitrogen gas at a heating rate of 20 °C/min.The raw and NaOH pretreated bamboo were hydrolyzed by cellulase in tubes with 3 mL of 50 mM sodium citrate buffer (pH 5.0) at 50 °C in a shaking incubator at 200 rpm. The dry matter (DM) content of the substrate was 2%. A 0.02% NaN3 was added to the hydrolysis broth to prevent bacterial infection. The cellulase preparation (CEL) contained both the Celluclast 1.5 L and Novozyme 188 preparations; the CEL was dosed at 5–40 FPU/g DM Celluclast 1.5 Land 500 nkat/g DM Novozyme 188. The samples were hydrolyzed, and then boiled for 10 min to stop enzymatic hydrolysis. After cooling, the samples were centrifuged at 10,000 rpm for 10 min, and the reducing sugars in the supernatants were analyzed. To investigate the synergistic interaction between CEL and XYL, hydrolysis of the NaOH pretreated bamboo by CEL and/or XYL was investigated as described above. CEL was dosed at 5 and 10 FPU Celluclast 1.5 L/g DM, and 500 nkat Novozyme 188/g DM. The XYL dosage was 2 mg protein/g DM. The samples were withdrawn after 48 h and boiled for 10 min to stop enzymatic hydrolysis. 2SO4 as the eluent at a flow rate of 0.5 mL/min. Before injection, the samples were filtered through 0.22-µm Micro-PES filters, and a volume of 20 µL was injected. Peaks were detected by refractive index and were identified and quantified by comparison to the retention times of authenticated standards (D-glucose and D-xylose).The concentration of glucose and xylose in the supernatants was determined using the Agilent 1260 infinity high performance liquid chromatography system . This system was equipped with a refractive index detector and an autosampler. An ion-moderated partition chromatography column (Aminex column HPX-87H) with a Cation H micro-guard cartridge was used. The column was maintained at 45 °C with 5-mM HThe glucose and xylose yields from the hydrolysis of samples were calculated using the following equations:2SO4 and glycerol pretreatments. In addition, the higher pretreatment temperature resulted in higher holocellulose and cellulose and lower lignin content. These results are in good agreement with previous studies reporting that an alkaline  pretreatment is effective for delignification of pinecones and 72% holocellulose was generated. The quantity of lignin decreased after pretreatment, and the order of lignin content was glycerol > H2SO4 > NaOH pretreatment. Relatively low lignin content may have occurred after the NaOH pretreatment because the effective lignin glass transition efficiently solubilized lignin, which was in accordance with a previous result that total lignin content decreases to 22.4%, with 13.7% acid soluble lignin and 8.7% Klason lignin after a dilute acid pretreatment. The acid and alkali pretreatments decreased the ash content but not the glycerol pretreatment. The large amount of ash after glycerol pretreatment may represent some ash and non-structural carbohydrates, starch, wax, including fat in glycerin, tannins, and pigments [The chemical composition of the raw material and pretreated bamboo samples is summarized in pigments .2SO4 pretreatment resulted in the highest DP value (372), followed by glycerol (362) and the NaOH pretreatment (329). The lowest DP value after the NaOH pretreatment was mainly due to deformation of the cellulose lattice during alkaline processing. The higher pretreatment temperature caused lower DP values for both the H2SO4 and NaOH pretreatments.The viscosities of the samples were determined and the related parameters were calculated to investigate the cellulose depolymerization behavior . The η a−1 range and the changes in the adsorption peaks of the functional groups are shown in −1 (characteristic peak of β-D glycosidase) in the pretreated bamboo samples was sharper than that in the untreated bamboo, indicating increased polysaccharide content after pretreatment [−1 was more obvious after the NaOH pretreatment than the bands after the H2SO4 and glycerol pretreatments, indicating higher polysaccharide contents in the NaOH pretreated bamboo. These results were in accordance with previous data  and hydrogen bond intensity (HBI), respectively. In general, the LOI is an empirical “crystallinity index”. The change in the LOI reflects the crystalline and amorphous regions. HBI reflects the crystalline cellulose and intra and intermolecular regularity [2SO4 and NaOH pretreated samples increased and a higher temperature gave rise to higher LOI values, indicating that the CrI values of bamboo increased after the H2SO4 and NaOH pretreatments, and the variation in the HBI values was opposite that of the LOI values. This study used the FT-IR spectroscopy results for the ratio of the absorption bands at 1428 cmgularity . Crystal2SO4 > NaOH, showing that the crystal layer spacing of cellulose after pretreatment decreased and the purity of cellulose increased. All samples exhibited typical cellulose I diffraction angles (2θ) of 16.02, 22.5, and 34.76°, which were assigned to the diffraction planes of 101, 002, and 040, respectively  were selected to compare the changes in chemical structure , C-3/5(non-ether-linked), C-1, and C-5 in guaiacyl units, respectively. The signals at 160.3 and 153.4 were assigned to C-3/5 (ether-linked) and C-1 in syringyl units, respectively. The reduction in these signals suggested that the aromatic groups of lignin in the pretreated bamboo had decreased significantly. Furthermore, the signal at 148.2 ppm in the NaOH pretreated bamboo was weaker than that of the Hfication .The signal at 89.2 ppm corresponded to C-4 of crystalline cellulose and became stronger after the pretreatment, particularly the NaOH pretreatment, indicating that the cellulose content in the pretreated bamboo samples had clearly increased. The signal at 84.6 ppm was divided into two peaks of 88.7 and 83.1 ppm for the NaOH pretreated native bamboo b, and th2SO4, and the NaOH pretreated bamboo, respectively. The increase in CrI after the pretreatments suggested the removal of lignin, hemicelluloses, and amorphous cellulose, which was consistent with the FT-IR and XRD results. Similar findings have been reported previously [As shown in eviously .2SO4 and NaOH pretreatments decreased from more than 50 ± 5 µm to 20 ± 2 µm (H2SO4) and less than 5 ± 2 µm (NaOH), indicating that NaOH effectively disrupted the robust structure of the raw materials relative to H2SO4. It is well known that lignin acts as a “glue” to strengthen cell walls. Cell walls will partly collapse without lignin, a highly branched and complex polymer with aromatic constituents [SEM micrographs of the untreated and pretreated bamboo samples are shown in tituents . NaOH ditituents .Therefortituents ,34.The TG and DTG curves of the native materials and pretreated samples are illustrated in 2SO4. The highest T2 (346 °C) in the NaOH pretreated sample showed remarkable thermal stability [All samples lost weight initially in the middle graded stage because of the evaporation of lower molecular weight compounds and loosely bound moisture at temperatures ≤ 100 °C ,7,17,35.est T2 34 °C in thThe hydrolysis yield of cellulose in native bamboo and pretreated bamboo (117 °C and 135 °C) by 1 mg protein/g DM XYL was 2.51–43.3%, indicating that some cellulolytic activities were present in the commercial XYL preparation, particularly in the 135 °C pretreated bamboo. After supplementing with 0.5 CEL to 1 mg protein/g DM XYL, the hydrolysis yield of NaOH pretreated bamboo at 135 °C increased from 84.2% to 98.1%. The hydrolysis yield of cellulose in NaOH pretreated bamboo further increased to 100.1%when using 1mg protein/g DM XYL with CEL. These results indicate that XYL supplementation was more effective than increasing cellulase loading during hydrolysis of the NaOH pretreated bamboo. The XYL preparation hydrolyzed residual xylan in the NaOH pretreated bamboo and increased the accessibility of cellulase to cellulose, which was consistent with a previous report regarding the synergy between CEL and XYL on the hydrolysis of steam pretreated corn stover and hydrothermally pretreated wheat straw .The reducing sugars released by the NaOH pretreated bamboo samples increased to different degrees with increasing dosages of CEL. These results were in good agreement with previous studies ,37.2SO4, and glycerol. The structural and chemical analysis results revealed that the NaOH pretreatment using a sand bath was the most effective method to compare H2SO4 with glycerol. The NaOH pretreatment resulted in uniform delignification, defibrillation, maximum CrI, and preferable thermo-stability. Decreases in diameter of the cellulose fibers from 50 ± 5 to 5 ± 2 µm and a DP of cellulose from 396 to 329 were observed. Supplementing with xylanase was more effective than increasing cellulase loading for scarification of the pretreated bamboo fractions, particularly after the NaOH pretreatment, demonstrating the positive effect of the NaOH pretreated bamboo in a sand bath with added xylanase for improving hydrolysis of bamboo. We carried out a comparative study to pretreat bamboo with NaOH, H


Honeysuckle has antiviral, antioxidative and anti-inflammatory properties. Allergic rhinitis (AR) is induced by immunoglobulin E (IgE)-mediated inflammatory reaction. Our study investigates whether honeysuckle extract (HE) has therapeutic effect on AR. An AR model of mice was established by ovalbumin (OVA). Hematoxylin–Eosin staining was used to assess nasal mucosa damage. Enzyme-linked immunosorbent assay (ELISA) was performed to determine serum histamine, IgE and interleukin (IL)-2, IL-4, IL-17 and interferon-γ (IFN-γ) from nasal lavage fluid. Western blot was carried out to analyze the protein level from nasal mucosa tissue. We found that HE not only decreased nasal rubbing and sneezing in AR mice, but also reduced AR-induced damage to nasal mucosa. Moreover, HE lowered the levels of serum IgE and histamine and inhibited IL-4 and IL-17 levels from AR mice but raised IL-2 and IFN-γ levels in AR-induced nasal lavage fluid. Our results also showed that HE elevated the protein levels of forkhead box P3 (Foxp3) and T-box transcription factor (T-bet) in AR-induced nasal mucosa tissue, whereas it inhibited signal transducer and activator of transcription (STAT) 3 and GATA binding protein 3 (GATA-3) protein levels. By regulating AR-induced inflammatory reaction and autoimmune response, HE also relieved OVA-induced AR. Thus, HE could be used as a potential drug to treat AR. Caused by inhaled allergens, allergic rhinitis (AR) is a hypersensitivity reaction-induced, immunoglobulin E (IgE)-mediated inflammatory reaction in nasal mucosa ,2. It isStrategies for treating AR were, for example, immunotherapy (pharmacologic) and elimination or avoidance of allergen (non-pharmacologic) ,7. PharmHE was purchased directly from Meiherb . HE was dissolved in saline solution  to form 50 mg/ml. The concentrations of HE25 and HE50 were 25mg/ml and 50mg/ml in the following experiments.n=8), while mice made as AR model were injected with 0.5 ml mixed solution containing ovalbumin   and aluminium hydroxide (20 mg/ml)  by intraperitoneal injection (ip) and fed for 7 and 14 days. OVA and aluminium hydroxide were dissolved in physiological saline , and the solution was sterilized by filtration . Mice were divided into four groups (n=8), which were AR group, Loratadine group, HE 25 group and HE50 group. At the 15th day, local stimulation was performed on all mice by adding OVA  into double nasal cavity during 10 days. In Loratadine group, mice were administrated Loratadine (2 mg/kg)  by irrigation before receiving daily local stimulation. In HE25 group, mice were administrated HE (25 mg/kg)  by irrigation before receiving daily local stimulation, while mice in HE50 group were administrated HE (50 mg/kg) by irrigation. All mice were placed under 12-h lighting (7:00–19:00) and 12-h dark (19:00–7:00) cycle in an air environment . Animal experimental operations in our study were performed in accordance with Guide for Animal Care and Use of Guangzhou Hospital of Integrated Traditional and Western Medicine. Animal experiments were approved by Ethics Committees and Health Authorities of Guangzhou Hospital of Integrated Traditional and Western Medicine (No. TW201803019E). The mice models of AR and the AR improvement by HE were evaluated for physiological and biochemical parameters [Male Balb/c mice aged 6 weeks were obtained from Animal Centre of Sun Yat-sen University  and were used to establish an AR model. Untreated mice  were regarded as control group . In addition, we could observe the eosinophils (EOS) infiltration. The EOS was observed as often being round or elliptical in shape, as well as having a double-leaf nucleus .Blood was extracted from mice after they were killed, and serum was obtained by centrifuging the blood collected . Histamine and IgE were determined by using histamine and IgE Enzyme-linked immunosorbent assay (ELISA) kit  according to the instructions.Nasal lavage fluid samples were collected from mice by washing their noses at the end of animal experiment. IL-2, IL-4, IL-17 and IFN-γ from nasal lavage fluid were detected by IL-2, IL-4, IL-17 and IFN-γ ELISA kit , respectively.Nasal mucosa was extracted from mice after the mice had been executed by decapitation. Nasal mucosa was fixed to paraffin section, dehydrated and paraffin-embedded. Hematoxylin dye  was used to stain the sections for 5 min, and the section was then washed by water. Next, ammonia was used to soak the sections, which were then washed by water. The sections were stained by 1% Eosin dye  and then washed by water.Nasal mucosa tissues were cut and centrifuged to extract proteins using TRIzol reagent . BCA kit  was used to measure protein concentration, and protein ladder  was separated from protein by SDS/PAGE. Next, the proteins were transferred to PVDF membranes , which were blocked by 5% albumin. The protein membranes were incubated first with primary antibody  was usedRNAs were extracted from nasal mucosa tissue by RNA purification kit . cDNAs were synthesized by extracted RNAs using reverse transcription kit . RNAs were amplified using cDNAs, primers  and tranP<0.05 indicated a significant difference.Western blot, Hematoxylin–Eosin staining, ELISA and quantitative polymerase chain reaction (qPCR) were performed for at least three times. All data were analyzed by F test using SPSS 19.0  and shown as Mean ± Standard Deviation (SD). AR significantly increased nasal rubbing A and sneP<0.01).AR significantly raised the levels of serum IgE A and hisP<0.01).AR lowered IL-2 A and IFNMice in the control group had hardly tissue edema, small vessel dilatation and gland hyperplasia, with a small amount of EOS infiltration, which was considered to be nasal damage. Compared with AR which caused seriously nasal damage  we foundP<0.01 or P<0.05).HE and Loratadine affected IL-17 level nasal mucosa tissue of AR mice A, which In our study, HE relieved the symptoms of AR such as sneezing and nasal itching, suggesting that HE had therapeutic effect on AR. Besides, a higher concentration of HE had a better effect on AR. AR patients tend to have a higher IgE in response to allergens, according to etiopathogenesis of AR . The cauAR model of mice was observed to have inflammation , and patFCRL3 [Allergen can cause autoimmune disease such as scleroderma, arthritis and rhinitis ,41. ImmuFCRL3 . T-bet eFCRL3 . HoweverFCRL3 . MoreoveFCRL3 . T-bet, FCRL3 . Innate FCRL3 . In the There are still some limitations, for example, the lack of more comprehensive scoring criteria and histological evaluation in this mouse model of AR and lacking of immunohistochemical analysis of signaling pathway proteins changes in nasal mucosa. We are planning to take out a further in-depth study.In conclusion, HE had a therapeutic efficacy for treating AR by assessing the symptoms of AR in AR model of mice. Moreover, HE relieved AR by decreasing AR-induced inflammation and autoimmunity. Thus, our study provided basic evidence that HE could be used as a drug for AR.


O-Fmoc-substituted benzyl orthoester derivative 2a was carried out in moderate yields by electrogenerated acid (EGA). The Fmoc group was effectively removed under mild basic conditions, and the product was submitted to the subsequent glycosylation.Tandem glycosylation of the 6- ESI-MS: calcd for C42H33O10NaBr 799.1155 (M+Na)+, found m/z 799.1151.A mixture of the crude and FmocCl  in pyridine (100 mL) was stirred overnight. The reaction was quenched by sat. aq. NaHCO-5-(((((9H-Fluoren-9-yl)methoxy)carbonyl)oxy)methyl)-2-((2-((tert-butyldimethylsilyl)oxy)-4-methoxybenzyl)oxy)-2-phenyltetrahydro-3aH-dioxolopyran-6,7-diyl dibenzoate (2a) and  2-tert-Buthyldimethylsyloxy-4-methoxyphenylmethyl2,3,4-tetra-O-benzoyl-6-O-9-fluorenylmethyloxycarboxyl-β-d-glucopyranoside (2b). To a solution of 5  in anhydrous toluene (0.9 mL) were added 6 , Hg(CN)2 , and MS 4A at 100 °C, and the mixture was stirred overnight. The mixture was filtered through a Celite pad, and the filtrate was washed with sat. aq. NaHCO3. The organic extracts were dried (Na2SO4), and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/acetone = 7:1) to give 2a  and 2b as oil: 2a: [α]23D +1.2 ; IR (film) 1728, 1613, 1257 cm−1; 1H-NMR (400 MHz) δ 0.15 , 0.93 , 3.74 , 4.16 , 4.29 , 4.78 , 5.37 , 5.76 , 6.04 , 6.31 , 6.48 , 7.18 , 7.41 , 7.57 , 7.75 , 7.82 , 7.97 , 8.11 ; 13C-NMR (100 MHz) δ −4.3 (SiCH3), 18.1 (tBu), 25.6 (tBu), 46.6 (Fmoc), 55.2 (OCH3), 61.3 (ArCH2), 67.3 (C-6), 68.3 (C-2), 69.2 (C-3), 70.1 (C-4), 72.3 (FmocCH2), 77.2 (C-5), 97.4 (C-1), 105.3 (Ar), 105.9 (Ar), 119.9 (Ar), 120.5 (Ar), 121.4 (Ar), 125.3 (orthoester-C), 126.5 (Ar), 127.1 (Ar), 127.2 (Ar), 127.8 (Ar), 128.3 (Ar), 1283.4 (Ar), 128.5 (Ar), 129.1 (Ar), 129.6 (Ar), 129.9 (Ar), 130.0 (Ar), 133.5 (Ar), 133.6 (Ar), 135.3 (Ar), 141.2 (Ar), 143.2 (Ar), 154.0 (C=O), 154.9 (C=O), 159.9 (C=O), 165.2 (C=O). ESI-MS: calcd for C56H56O13SiNa 987.3388 (M+Na)+, found m/z 987.3373. 2b: [α]23D −14.9 ; IR (film) 1735, 1610, 1262 cm−1; 1H-NMR (400 MHz) δ 0.10 , 0.16 , 0.94 , 3.72 , 4.06 , 4.23 , 4.40 , 4.76 , 4.83 , 4.86 , 5.58 , 5.60 , 5.83 , 6.27 , 6.30 , 7.12 , 7.35 , 7.50 , 7.62 , 7.76 , 7.83 , 7.92 ; 13C-NMR (100 MHz) δ −4.4 (SiCH3), −4.3 (SiCH3), 18.1 (tBu), 25.6 (tBu), 29.7 (tBu), 46.6 (Fmoc), 55.2 OCH3), 65.6 (ArCH2), 66.3 (C-6), 69.6 (C-4), 70.2 (C-3), 71.6 (C-2), 72.0 (C-5), 72.9 (FmocCH2), 99.1 (C-1), 105.4 (Ar), 105.9 (Ar), 119.6 (Ar), 119.9 (Ar), 125.2 (Ar), 125.3 (Ar), 127.2 (Ar), 127.8 (Ar), 128.1 (Ar), 128.2 (Ar), 128.4 (Ar), 128.8 (Ar), 129.3 (Ar), 129.7 (Ar), 129.8 (Ar), 129.9 (Ar), 131.2 (Ar), 133.0 (Ar), 133.2 (Ar), 133.4 (Ar), 141.2 (Ar), 143.2 (Ar), 143.4 (Ar), 154.7 (C=O), 154.9 (C=O), 160.2 (C=O), 164.9 (C=O), 165.2 (C=O), 165.8 (C=O). ESI-MS: calcd for C56H56O13SiNa 987.3388 (M+Na)+, found m/z 987.3381.-2-((2-((tert-Butyldimethylsilyl)oxy)-4-methoxybenzyl)oxy)-5-(hydroxymethyl)-2-phenyl-tetrahydro-3aH-dioxolopyran-6,7-diyl dibenzoate (3). To a solution of 2a  in pyridine (0.5 mL) was added Et3N  at room temperature. After being stirred overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/acetone = 2:1) to give 3 as a clear oil : [α]23D +9.5 ; IR (film) 3527, 1725, 1613, 1261 cm−1; 1H-NMR (400 MHz) δ 0.15 , 0.90 , 3.65 , 3.75 , 3.78 , 3.89 ,4.29 , 4.36 , 4.75 , 5.37 , 5.74 , 6.03 , 6.31 , 6.48 , 7.18 , 7.44 , 7.60 , 7.81 , 7.98 , 8.05 ; 13C-NMR (100 MHz) δ −4.3 (SiCH3), 18.1 (tBu), 25.6 (tBu), 55.2 (OCH3), 61.2 (ArCH2), 62.4 (C-6), 68.3 (C-2), 69.4 (C-3), 69.8 (C-4), 72.6 (C-5), 97.5 (C-1), 105.3 (Ar), 105.9 (Ar), 120.5 (Ar), 121.1 (Ar), 126.4 (orthoester-C), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 129.0 (Ar), 129.1 (Ar), 129.6 (Ar), 129.9 (Ar), 130.0 (Ar), 133.5 (Ar), 133.6 (Ar), 135.3 (Ar), 154.0 (C=O), 159.9 (C=O), 164.6 (C=O), 165.6 (C=O). ESI-MS: calcd for C41H46O11NaSi 765.2707 (M+Na)+, found m/z 765.2710.Cyclohexyl 2,3,4-tri-O-benzoyl-6-O-9-fluorenylmethyloxycarboxyl-β-d-glucopyranoside (7). A 0.1 M solution of cyclohexanol in 1,2-DCE (10 mL) containing 4Å MS and Bu4NClO4  as a supporting salt, was electrolyzed by constant current electrolysis  at 40 °C, using a glassy carbon plate (1.5 cm × 1.5 cm) as an anode and a Pt plate (1.8 cm × 1.8 cm) as a cathode. After the electrolysis, the reaction mixture  was added to a solution of 2a , cyclohexanol , and 4Å MS in 1,2-DCE (1.1 mL). After being stirred 20 min, the mixture was filtered through a Celite pad and the filtrate was washed with sat. aq. NaHCO3. The organic extracts were dried (Na2SO4), and concentrated in vacuo. The residue was purified by preparative TLC (hexane/acetone = 5:1) to give 7 as an oil : [α]23D −4.10 ; IR (film) 1732, 1601, 1262 cm−1; 1H-NMR (400 MHz) δ 1.22 , 1.46 , 1.70 , 1.88 , 3.72 , 4.09 , 4.22 , 4.36 , 4.46 , 4.93 , 5.51 , 5.55 , 5.90 , 7.30 , 7.51 , 7.60 , 7.77 , 7.84 , 7.95 ; 13C-NMR (100 MHz) δ 23.4 (cyclohexyl), 23.6 (cyclohexyl), 25.3 (cyclohexyl), 31.5 (cyclohexyl), 33.1 (cyclohexyl), 46.6 (Fmoc), 66.5 (C-6), 69.8 (C-4), 70.2 , 72.0 (FmocCH2), 72.9 (C-5), 78.2 (cyclohexyl), 99.7 (C-1), 120.0 (Ar), 125.2 (Ar), 127.2 (Ar), 127.9 (Ar), 128.3 (Ar), 128.4 (Ar), 128.7 (Ar), 128.8 (Ar), 129.5 (Ar), 129.7 (Ar), 129.8 (Ar), 133.1 (Ar), 133.2 (Ar), 133.5 (Ar), 141.2 (Ar), 143.2 (Ar), 123.3 (Ar), 154.8 (C=O), 164.9 (C=O), 165.3 (C=O), 165.8 (C=O). ESI-MS: calcd for C48H45O117997.2962 (M+H)+, found m/z 797.2936.Cyclohexyl 2,3,4-tri-O-benzoyl-β-d-glucopyranoside (8). To a solution of 7  in pyridine (1 mL) was added Et3N  at room temperature, and the mixture was stirred overnight. The mixture was concentrated invacuo. The residue was purified by silica gel column chromatography (hexane/acetone = 2:1) to give 8 as an oil : [α]23D −11.5 ; IR (film) 3523, 1731, 1601, 1263 cm−1; 1H-NMR (400 MHz) δ 1.05 , 1.48 , 1.77 , 2.41 , 3.68 , 4.38 , 5.37 , 5.82 , 7.18 , 7.31 , 7.41 , 7.74 , 7.85 ; 13C-NMR (100 MHz) δ 23.4 (cyclohexyl), 23.6 (cyclohexyl), 25.4 (cyclohexyl), 31.5 (cyclohexyl), 33.2 (cyclohexyl), 61.5 (C-6), 69.7 , 72.1 , 72.9 , 74.5 (C-5), 78.1 (cyclohexyl), 99.6 , 128.2 (Ar), 128.3 (Ar), 128.5 (Ar), 128.6 (Ar), 128.9 (Ar), 129.5 (Ar), 129.6 (Ar), 129.7 (Ar), 129.9 (Ar), 133.1 (Ar), 133.2 (Ar), 133.6 (Ar), 164.9 (C=O), 165.8 (C=O), 165.9 (C=O). ESI-MS: calcd for C33H34O9Na 597.2101 (M+Na)+, found m/z 597.2112.d-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-6-O-9-fluorenyl-methyloxycarboxyl-β-d-glucopyranoside (9). A solution of cyclohexanol in 1,2-DCE  containing 4Å MS and Bu4NClO4  as a supporting salt, was electrolyzed by constant current electrolysis  at 40 °C, using a glassy carbon plate (1.5 cm × 1.5 cm) as an anode and a Pt plate (1.8 cm × 1.8 cm) as a cathode. The reaction mixture  was added to a solution of 2a  and 8 , and MS 4A in 1,2-DCE (0.5 mL). After being stirred 20 min, the mixture was filtered through a Celite pad, and the filtrate was washed with sat. aq. NaHCO3. The organic extract was dried (Na2SO4), and concentrated in vacuo. The residue was purified by preparative TLC (hexane/acetone = 2:1) to give 9 as an oil : [α]23D −9.8 ; IR (film) 1734, 1601, 1261 cm−1; 1H-NMR (400 MHz) δ 1.41 , 3.62 , 4.00 , 4.29 , 4.78 , 5.04 , 5.34 , 5.41 , 5.48 , 5.52 , 5.80 , 5.83 , 7.41 , 7.62 , 7.78 , 7.92 ; 13C-NMR (100 MHz) δ 22.9 (cyclohexyl), 23.2 (cyclohexyl), 25.4 (cyclohexyl), 31.2 (cyclohexyl), 32.9 (cyclohexyl), 46.6 (Fmoc), 66.0 (C-6), 68.3 (C-6'), 69.3(C-4), 69.9 (C-4'), 70.3 (C-3), 71.7 , 71.9 (C-2'), 72.1 C-5'), 72.8 (C-5), 72.9 (cyclohexyl), 74.1 (FmocCH2), 99.4 (C-1'), 100.8 (C-1), 119.9 (Ar), 125.3 (Ar), 125.4 (Ar), 127.2 (Ar), 127.8 (Ar), 128.2 (Ar), 128.3 (Ar), 128.4 (Ar), 128.7 (Ar), 128.9 (Ar), 129.3 (Ar), 129.5 (Ar), 129.6 (Ar), 129.7 (Ar), 129.8 (Ar), 133.0 (Ar), 133.1 (Ar), 133.2 (Ar), 133.4 (Ar), 133.5 (Ar), 141.2 (Ar), 143.3 (Ar), 143.4 (Ar), 154.8 (C=O), 164.9 (C=O), 165.1 (C=O), 165.2 (C=O), 165.4 (C=O), 165.6 (C=O), 165.7 (C=O). ESI-MS: calcd for C75H66O19Na 1293.4096 (M+Na)+, found m/z 1293.4078.Cyclohexyl 2,3,4-tri-O-benzoyl-β-Phenyl 2,3,4-tri-O-benzoyl-6-O-9-fluorenylmethyloxycarboxyld-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-d-glucopyranoside-β- (11). A solution of cyclohexanol in 1,2-DCE  containing 4Å MS and Bu4NClO4  as a supporting salt was electrolyzed by constant current electrolysis  at 40 °C, using a glassy carbon plate (1.5 cm × 1.5 cm) as an anode and a Pt plate (1.8 cm × 1.8 cm) as a cathode. After the electrolysis, the reaction mixture  was added to a solution of 2a , 10 , m and 4Å MS in 1,2-DCE (0.5 mL). After being stirred for 20 min, the reaction mixture was filtered through a Celite pad, and the filtrate was washed with sat. aq. NaHCO3. The organic extracts were dried (Na2SO4), and concentrated in vacuo. The residue was purified by preparative TLC (hexane/acetone = 2:1) to give 11 as an oil : [α]23D +8.9 ; IR (film) 1733, 1601, 1261 cm−1; 1H-NMR (400 MHz) δ 3.99 , 4.24 , 4.38 , 4.92 , 4.97 , 5.31 , 5.34 , 5.51 , 5.81 , 5.85 , 7.23 , 7.39 , 7.61 , 7.74 , 7.85 , 7.93 ; 13C-NMR (100 MHz) δ 46.6 (Fmoc), 65.9 (C-6), 68.4 (C-6'), 69.4 (C-4'), 69.5 (C-4'), 70.2 (C-3'), 70.4 (C-3), 71.6 (C-2), 72.0 (C-2'), 72.8 (C-5'),74.0 (FmocCH2), 78.3 (C-5), 85.9 (C-1'), 101.0 (C-1), 119.9 (Ar), 125.3 (Ar), 125.4 (Ar), 127.2 (Ar), 128.2 (Ar), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 128.6 (Ar), 128.7 (Ar), 128.8 (Ar), 129.1 (Ar), 129.2 (Ar), 129.7 (Ar), 129.8 (Ar), 131.7 (Ar), 133.2 (Ar), 133.4 (Ar), 133.5 (Ar), 141.2 (Ar), 141.3 (Ar), 143.3 (Ar), 143.4 (Ar), 154.8 (C=O), 164.9 (C=O), 165.1 (C=O), 165.2 (C=O), 165.3 (C=O), 165.6 (C=O), 165.7 (C=O). ESI-MS: calcd for C75H60O18NaS 1303.3398 (M+Na)+, found m/z 1303.3385.Phenyl 2,3,4-tri-O-benzoyl-6-O-9-fluorenylmethyloxycarboxyl-βd-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl--βd-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio--βd-glucopyranoside- (12). To a solution of 11  in pyridine (0.5 mL) was added Et3N  at room temperature. After being stirred overnight, the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/acetone = 2:1) to give an alcohol as an oil : [α]23D −2.1 ; IR (film) 3062, 1731, 1601, 1262 cm−1; 1H-NMR (400 MHz) δ 2.81 , 3.60 , 3.74 , 3.95 , 4.05 , 4.92 , 5.23 , 5.40 , 5.81 , 5.85 , 7.38 , 7.53 , 7.76 , 7.81 , 7.93 ; 13C-NMR (100 MHz) δ 61.2 (C-6), 67.9 (C-6'), 69.4 (C-4'), 69.9 (C-4), 70.4 (C-3'), 71.5 (C-3), 72.8 (C-2), 73.9 (C-2'), 74.6 (C-5'), 77.8 (C-5), 86.0 (C-1'), 100.5 (C-1), 128.2 (Ar), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 128.7 (Ar), 128.8 (Ar), 129.1 (Ar), 129.2 (Ar), 129.7 (Ar), 129.8 (Ar), 129.9 (Ar), 131.5 (Ar), 133.2 (Ar), 133.3 (Ar), 133.5 (Ar), 133.6 (Ar), 164.9 (C=O), 165.1 (C=O), 165.6 (C=O), 165.7 (C=O), 165.9 (C=O). ESI-MS: calcd for C60H50O16NaS 1081.2717 (M+Na)+, found m/z 1081.2732.4NClO4  as a supporting salt, was electrolyzed by constant current electrolysis  at 40 °C, using a glassy carbon plate (1.5 cm × 1.5 cm) as an anode and a Pt plate (1.8 cm × 1.8 cm) as a cathode. The reaction mixture  was added to a solution of 2a , the alcohol , and 4Å MS in 1,2-DCE (0.5 mL). After being stirred at 40 °C for 20 min, the reaction mixture was filtered through a Celite pad, and the filtrate was washed with saturated aqueous NaHCO3. The organic extracts were dried (Na2SO4), and concentrated in vacuo. The residue was purified by preparative TLC (Et2O/hexane = 2:1) to give 12 as a clear oil : [α]23D −5.4 ; IR (film) 1733, 1601, 1261 cm−1; 1H-NMR (400 MHz) δ 3.62 , 3.84 , 4.01 , 4.31 , 4.61 , 4.99 , 5.08 , 5.10 , 5.21 , 5.51 , 5.68 , 5.84 , 6.06 , 7.18 , 7.36 , 7.58 , 7.76 , 7.92 ; 13C-NMR (100 MHz) δ 46.6 (Fmoc), 66.2 (C-6), 68.3 (C-6''), 69.4 (C-6'), 69.7 (C-4''), 70.1 (C-4), 70.2 (C-4'), 70.5 (C-3''), 71.7 (C-3), 71.9 , 72.0 (C-2''), 72.6 (C-5'), 72.7 (C-5''), 73.9 (C-5), 74.1 ((FmocCH2), 86.3 (C-1''), 100.5 (C-1), 101.2 (C-1'), 119.9 (Ar), 120.0 (Ar), 125.3 (Ar), 125.4 (Ar), 127.2 (Ar), 127.8 (Ar), 128.1 (Ar), 128.2 (Ar), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 128.6 (Ar), 128.7 (Ar), 128.8 (Ar), 129.0 (Ar), 129.1 (Ar), 129.3 (Ar), 129.4 (Ar), 129.7 (Ar), 129.8 (Ar), 130.0 (Ar), 132.1 (Ar), 132.8 (Ar), 133.0 (Ar), 133.2 (Ar), 133.4 (Ar), 141.2 (Ar), 143.3 (Ar), 143.4 (Ar), 154.8 (C=O), 164.9 (C=O), 165.2 (C=O), 165.3 (C=O), 165.7 (C=O). ESI-MS: calcd for C102H82O26NaS 1777.4713 (M+Na)+, found m/z 1777.4736.A solution of cyclohexanol in 1,2-DCE  containing 4Å MS and Bu2a bearing the selectively removable Fmoc group at the C-6 position was synthesized, and its utility as a glycosyl donor and acceptor after deprotection of the Fmoc group was demonstrated.The orthoester


Treating potassium ferrocenyldithiofluorophosphinic acid with an equimolar amount of tetraphenylphosphonium chloride readily yielded the corresponding organic adducts, and with mono- and di-halogenated alkanes generated a series of the corresponding esters of ferrocenylphosphonofluoridodithioates. Similarly, using 1,3-epithionaphthooxadiphosphinine 1,3-disulfide or Belleau’s Reagent in place of FcLR resulted in the corresponding novel salts, adducts, and ester derivatives. All new compounds have been characterized by means of multi-NMR  spectroscopy and accurate mass measurement in conjunction with single crystal X-ray crystallography of four structures.A series of novel ferrocenyl- and aryl-phosphonofluorodithioic salts, adducts, and esters has been prepared. The reaction of 2,4-diferrocenyl-1,3,2,4-diathiadiphosphetane 2,4-disulfide {[FcP(μ-S)S] Sincessively ,17,18,19rus atom ,20,21. Tde (TAF) ,23. Simide (TAF) . The impde (TAF) . Herein,2, FcLR} has been reported by our group ur group ,27. FcLR9% yield . Both re1 was obtained as a yellow solid and is insoluble in organic solvents but soluble in oxygen-free water and slowly decomposed when stored at room temperature. Organic salt 2 was prepared as golden sticky oil, is soluble in organic solvents, and shows good air stability at room temperature. Both compounds show the anticipated molecular ion peaks [M − K]− or [M – N(n-Bu)4]− and satisfactory accurate mass measurement. The 31P-NMR spectra exhibits doublets at δp = 132.1 ppm in compound 1 and 127.3 ppm in compound 2, respectively, attributable to the presence of the P–F single bond, and the values are significantly bigger than that in their selenium counterpart PSe2 ions +, and were confirmed by satisfactory accurate mass measurements. Not surprisingly, the 31P- and 19F-NMR spectra of 3 show similar patterns to 1 and 2 with identical 1J coupling constants apart from another singlet signal in the 31P-NMR spectrum at δP = 23.2 ppm, assigned to the PPh4 cation ion. The 31P-NMR spectra of 4–9 display signals ranging from δP = 116.0 to 118.0 ppm with 31P–19F coupling constants differing from J = 1098–1101 Hz. In the 19F-NMR spectra of compounds 4–9, two equal signals in the range δF = −42.6–−38.3 ppm being considerately bigger than that in their selenium counterpart esters (δF = −58.5–−55.8 ppm) [31P–19F coupling constants.Compounds 5.8 ppm)  are obsecd]oxadiphosphinine-1-thiolate 1,3-disulfide 11 and tetrabutylammonium, 3-fluoronaphthooxadiphosphinine-1-thiolate 1,3-disulfide salt 12 as shown in cd]oxadiphosphinine 1,3-disulfide 10 was obtained from the half-oxidization of the know compound 1,3-epithionaphthothiadiphosphinine 1,3-disulfide oxadiphosphinine-1-thiolate 1,3-disulfide 11 in 98% yield as a pale yellow solid; or with two equivalents of tetrabutylammonium fluoride (TBAF) at room temperature in tetrahydrofuran for 1 h gave tetrabutylammonium 3-fluoronaphthooxadiphosphinine-1(3H)-thiolate 1,3-disulfide 12 in 85% yield as a brown sticky paste. The P–O–P bond could not be broken even when an excess of potassium fluoride or tetrabutylammonium fluoride was used, indicating that the P–O–P bridge is more robust than the P–S–P bridge in the reaction.By using the same procedure, we have carried out the synthesis of potassium 3-fluoronaphthothiadiphosphinine-1-thiolate 1,3-disulfide salt (1144 Hz) − complexes [1.973(2)–2.046(2) Å] [ca. 2.10 Å] [ca. 1.91 Å] [2− fragment. The P–F bond length [1.615(4) Å] is statistically indistinguishable from that in PSe2F− [1.604(3)–1.610(5) Å] [3 as shown in Crystals of 46(2) Å] ,34; howe 2.10 Å]  and the  1.91 Å] , indicat10(5) Å] . The thr13 reveals that the naphthalene part of the molecule and two phosphorus atoms lie very close to the mean plane fitted to these atoms [maximum deviation 0.092 Å for C(1)], with the oxygen atom lying 0.548 Å above the mean plane and its cationic part being identical to that in 3. The structures of the cation in compounds 3 and 13 are noteworthy. The orientations of four phenyl ring are distorted away from each other, as evidenced by the different dihedral angles between three facing anion ion phenyl rings and one away phenyl ring  in the cation Ph4P+ ions. This distortion presumably arises as a result of steric interactions of the Ph4P+ cation ion with the anionic fragments FPS2− and C10H6P2S3OF−. The three-dimensional network in The X-ray structure of 14 has the same anionic part as that in the structure of 13. However, in contrast to 13, the C10H6P2 part of the anion is planar with the oxygen atom being significantly distorted and lying 0.495 [0.490] Å above the mean plane. The C3P2O ring is buckled with the C3P2 and P2O planes, inclined with respect to each other by 46.3° in 13 and 42.0° [44.1°] in 14. It is worth noting that the opening of the P2SO ring results in substantial lengthening of the transanular P···P distances {2.900 Å in 13 and 2.923 Å [2.927 Å] in 14vs. 2.73 Å} [13 [125.5(4)°] is significantly smaller than the corresponding values in 14 {128.0(4) [128.9(4)]°}, indicating the steric effect of the different cationic parts. However, the P–O–P angles are different from each other {125.5(4)° in 13 and 128.0(4) [128.9(4)]° in 14}; these values are much wider than in similar P–S–P structures [100.21(4)–102.69(4)°] [14 [1.563(8) Å] are slightly shorter than in 13 [1.593(8) Å], 2 [1.615(4) Å], and PSe2F− [1.604(3)–1.610(5) Å]  and the P=S double bond lengths [1.8939(12) Å] [13 [1.883(4) Å] and 14 {1.891(4) [1.819(5)] Å} are slightly shorter than that in PO3S systems [1.888(8)–1.8987(16) Å]  Å} is significantly longer than the other {1.573(6) or 1.577(8) [1.569(8)] Å}. The aggregation in The single crystal structure of compound  2.73 Å} ,37. The .69(4)°] ,35,37. S9(12) Å] . The P=S7(16) Å] , and two18 as shown in 2, 13 and 14. The naphthalene part of the molecule and two phosphorus atoms lies very close to a mean plane [maximum deviation 0.071 Å for P(3)]. The C3P2O is non-planar with a dihedral angle of 46.73° about P···P axis. The small newly formed six-membered P2S2CO ring is notable distorted from planar: atom O(2) lies 0.631 Å below and atom C(11) lies 0.915 Å above the P(1)–P(3)–S(4)–S(6) mean plane with two S(1) and S(3) atoms below the mean plane. The newly formed eight-membered C3P2S2C ring is hinged with the C3P2 and P2S2 mean planes inclined by 74.2° with respect to each other. The transanular P···P distance is 2.916 Å. The P(1)–S(1) [1.9020(13) Å] and P(3)–S(3) [1.9097(13) Å] and P(1)–S(6) [2.0762(14) Å] and P(3)–S(4) [2.0730(14) Å] distances are comparable to those in other known C3P2S ring derivatives  −. Accurate mass measurement (CI−MS): 298.9208 [C10H9FFePS2]−, calculated mass for [C10H9FFePS2]−: 298.9222.A mixture of 2,4-diferrocenyl-1,3,2,4-diathiadiphosphetane 2,4-disulfide  and tetrabutylammonium fluoride  in THF (50 mL) was heated at 80 °C for 1 h. Upon cooling to room temperature the reaction mixture was filtered to remove unreacted solid and the filtrate was evaporated in vacuo to give the title compound 2 as golden sticky oil . Selected IR : 1638 (m), 1467 (s), 1382 (m), 1172 (m), 1106 (m), 1023 (s), 884 (m), 822 (m), 738 (m), 698 (vs), 586 (m), 489 (s). 1H-NMR , 4.60–4.55 , 4.31–4.27 , 4.22 , 3.25–3.18 , 1.67–1.58 , 1.42–1.38 , 0.97  = 6.6 Hz, 12H, CH3) ppm. 13C-NMR , 72.5  = 67.6 Hz, ferrocenyl-C), 71.5  = 15.5 Hz, ferrocenyl-C), 70.6  = 17.7 Hz, ferrocenyl-C), 70.2 , 59.0, 24.1, 19.9, 13.6 ppm. 31P-NMR , 127.3  = 1009 Hz) pm. 19F-NMR , −14.0  = 1008 Hz) ppm. MS , 242 [C16H36N]+. Accurate mass measurement (CI+MS): 242.2840 [C16H36N]+, calculated mass for [C16H36N]+: 242.2846; MS , 299 [C10H9FFePS2]−. Accurate mass measurement (CI−MS): 298.9260 [C10H9FFePS2]−, calculated mass for [C10H9FFePS2]−: 298.9269.A suspension of in vacuo to give the title compound 3 as pale yellow solid . M.p. 175–176 °C. Selected IR : 1585 (m), 1483 (m), 1437 (s), 1168 (m), 1107 (s), 1021 (m), 997 (m), 817 (m), 758 (m), 721 (s), 690 (vs), 585 (m), 529 (s). 1H-NMR , 7.97–7.91 , 7.81–7.71 , 7.67–7.63 , 4.60–4.57 , 4.24 , 4.22–4.20  ppm. 13C-NMR , 136.6  = 3.0 Hz, Ar-C), 135.3  = 10.1 Hz, Ar-C), 131.5  = 12.7 Hz, Ar-C), 118.3  = 89.8 Hz, Ar-C), 72.3  = 15.2 Hz, ferrocenyl-C), 71.3  = 56.4 Hz, ferrocenyl-C), 70.9 , 70.4  = 13.1 Hz, ferrocenyl-C) ppm. 31P-NMR , 126.8  = 1010 Hz), 23.2 (s) pm. 19F-NMR , −14.5  = 1008 Hz) ppm. MS , 299 [C10H9FFePS2]−. Accurate mass measurement (CI−MS): 298.9210 [C10H9FFePS2]−, calculated mass for [C10H9FFePS2]−: 298.9222. MS , 339 [C24H20P]+. Accurate mass measurement (CIMS): 339.1299 [C24H20P]+, calculated mass for [C24H20P]+: 339.1297.Potassium ferrocenylphosphonofluoridodithioate  was dissolved in degassed water (10 mL). To this solution was added dropwise an equivalent of tetraphenylphosphonium chloride  in water (10 mL). The precipitation was almost immediately formed and the mixture was allowed to continue stirring for 1 h. The salt was harvested by filtration and was dried over 110 °C p-xylene dibromide . The mixture was stirred at room temperature for 24 h. After filtration to remove the unreacted solid and drying under reduced pressure, the residue was purified by column chromatography on silica gel using dichloromethane as eluent to give the title compound 4 as greenish yellow oil . Selected IR : 1492 (w), 1454 (w), 1412 (m), 1186 (s), 1107 (m), 1027 (s), 1002 (m), 820 (s), 764 (m), 700 (s), 607 (m), 548 (s), 488 (s). 1H-NMR , 7.36–7.27 , 4.70–4.60 , 4.57 , 4.37–4.31  ppm. 13C-NMR , 131.2 (Ar-C), 131.0 (Ar-C), 129.4 (Ar-C), 128.7 (Ar-C), 75.0  = 75.1 Hz, ferrocenyl-C), 72.6  = 13.5 Hz, ferrocenyl-C), 72.2  = 19.3 Hz, ferrocenyl-C), 70.8 , 31.0 ppm. 31P-NMR , 115.5  = 1103 Hz) pm. 19F-NMR , −39.6  = 1104 Hz) ppm. MS , 702 [C28H26F2Fe2P2S4]+. Accurate mass measurement (EI+MS): 701.9057 [C28H26F2Fe2P2S4]+, calculated mass for [C28H26F2Fe2P2S4]+: 701.9059.A mixture of tetrabutylammonium ferrocenylphosphonofluoridodithioate  in THF (30 mL) was added to 5 as pale yellow paste . Selected IR : 1604 (m), 1521 (s), 1347 (s), 1186 (m), 1108 (m), 1029 (m), 821 (m), 801 (m), 699 (s), 548 (m), 493 (m). 1H-NMR , 8.17  = 7.5 Hz, 2H, Ar-H), 7.56  = 7.5 Hz, 2H, Ar-H), 4.64–4.34 , 4.33  ppm. 13C-NMR , 144.9 (Ar-C), 130.0 (Ar-C), 129.4 (Ar-C), 123.8 (Ar-C), 72.7  = 13.6 Hz, ferrocenyl-C), 72.2  = 19.7 Hz, ferrocenyl-C), 71.1  = 14.5 Hz, ferrocenyl-C), 70.8 , 36.7 (SCH2) ppm. 31P-NMR , 116.0  = 1103 Hz) ppm. 19F-NMR , −38.8  = 1104 Hz) ppm. MS , 453 [C17H15FFeNO2PS2NH4]+. Accurate mass measurement (CI+MS): 451.0002 [C17H15F54FeNO2PS2NH4]+, calculated mass for [C17H15F54FeNO2PS2NH4]+: 451.0000.A mixture of tetrabutylammonium ferrocenylphosphonofluoridodithioate  in THF (30 mL) was added to 4-nitrobenzyl bromide . The mixture was stirred at room temperature for 24 h. After filtration to remove the unreacted solid and drying under reduced pressure, the residue was purified by column chromatography on silica gel using dichloromethane as eluent to give the title compound 6 as reddish yellow paste . Selected IR : 1468 (m), 1440 (m), 1412 (m), 1186 (s), 1107 (m), 1027 (s), 820 (s), 760 (m), 730 (m), 699 (vs), 548 (s), 489 (s). 1H-NMR , 7.57  = 8.0 Hz, Ar-H), 7.26  = 8.0Hz, Ar-H), 4.63–4.36 , 4.34  ppm. 13C-NMR , 133.1 (Ar-C), 131.4 (Ar-C), 129.6 (Ar-C), 127.9 (Ar-C), 72.3  = 13.4 Hz, ferrocenyl-C), 72.1  = 19.7 Hz, ferrocenyl-C), 71.3  = 15.6 Hz, ferrocenyl-C), 70.8 , 38.1 (SCH2) ppm. 31P-NMR , 116.4  = 1103 Hz) ppm. 19F-NMR , −38.3  = 1105 Hz) ppm. MS , 469 [C17H15BrFFePS2H]]+. Accurate mass measurement (CI+MS): 466.8988 [C17H15BrF54FePS2H], calculated mass for [C17H15BrF54FePS2H]+: 466.8989.A mixture of tetrabutylammonium ferrocenylphosphonofluoridodithioate  in THF (30 mL) was added to 4-bromobenzyl bromide . The mixture was stirred at room temperature for 24 h. After filtered to remove unreacted solid and dried under reduced pressure, the residue was purified by column chromatography on silica gel using dichloromethane as eluent to give the title compound 7 as red oil . Selected IR : 1413 (m), 1312 (m), 1186 (s), 1107 (m), 1028 (s), 1003 (w), 820 (vs), 699 (vs), 551 (s), 492 (s). 1H-NMR , 4.67–4.39 , 4.36 , 2.44  = 16.5 Hz, J = 1.7 Hz, 3H, SCH3) ppm. 13C-NMR , 72.5  = 12.6 Hz, ferrocenyl-C), 72.2  = 19.3 Hz, ferrocenyl-C), 70.8 , 14.6  = 4.2 Hz, SCH3) ppm. 31P-NMR , 118.0  = 1099 Hz) pm. 19F-NMR , −42.6  = 1102 Hz) ppm. MS , 315 [M + H]+. Accurate mass measurement (CI+MS): 312.9569 [M + H]+, calculated mass for [C11H12F54FePS2H]+: 312.9571.A mixture of tetrabutylammonium ferrocenylphosphonofluoridodithioate  in THF (30 mL) was added to iodomethane . The mixture was stirred at room temperature for 24 h. After filtration to remove the unreacted solid and drying under reduced pressure, the residue was purified by column chromatography on silica gel using dichloromethane as eluent to give the title compound 8 as pale golden paste . Selected IR : 2229 (s), 1484 (m), 1433 (m), 1411 (m), 1276 (m), 1186 (s), 1028 (m), 1002 (m), 905 (m), 823 (s), 804 (s), 701 (vs), 601 (m), 545 (s), 492 (s). 1H-NMR , 7.69–7.34 , 4.60–4.35 , 4.25 , 4.28–4.22  ppm. 13C-NMR , 133.6 (Ar-C), 132.9 (Ar-C), 132.6 (Ar-C), 132.1 (Ar-C), 131.4 (Ar-C), 129.7 (Ar-C), 72.6  = 13.6 Hz, ferrocenyl-C), 72.3  = 19.6 Hz, ferrocenyl-C), 71.1  = 15.4 Hz, ferrocenyl-C), 70.8 , 36.6 (SCH2) ppm. 31P-NMR , 116.1  = 1101 Hz), 116.0  = 1101 Hz) pm. 19F-NMR , −39.1  = 1103 Hz) ppm. MS , 433 [C18H15FFeNPS2NH4]+. Accurate mass measurement (CI+MS): 431.0101 [C18H15F54FeNPS2NH4]+, calculated mass for [C18H15F54FeNPS2NH4]+: 431.0102.A mixture of tetrabutylammonium ferrocenylphosphonofluoridodithioate  in THF (30 mL) was added to 4-cyanobenzyl bromide . The mixture was stirred at room temperature for 24 h. After filtration to remove the unreacted solid and driying under reduced pressure, the residue was purified by column chromatography on silica gel using dichloromethane as eluent to give the title compound 9 as reddish-yellow oil . Selected IR : 1493 (m), 1454 (m), 1412 (m), 1186 (s), 1107 (m), 1027 (s), 1002 (m), 820 (vs), 698 (vs), 548(s), 492 (s). 1H-NMR , 7.34–7.21 , 4.64–4.35 , 4.32 , 4.24–4.21  ppm. 13C-NMR , 137.0 (Ar-C), 129.1 (Ar-C), 128.8 (Ar-C), 127.8 (Ar-C), 72.9  = 11.6 Hz, ferrocenyl-C), 72.5  = 13.6 Hz, ferrocenyl-C), 72.4  = 19.7 Hz, ferrocenyl-C), 71.2  = 15.4 Hz, ferrocenyl-C), 70.8 , 37.5 (SCH2) ppm. 31P-NMR , 116.3  = 1101 Hz) pm. 19F-NMR , −39.1  = 1103 Hz) ppm. MS , 391 [C17H16FFePS2H]+. Accurate mass measurement (CI+MS): 388.9880 [C17H16F54FePS2H]+, calculated mass for [C17H16F54FePS2H]+: 388.9884.A mixture of tetrabutylammonium ferrocenylphosphonofluoridodithioate  in THF (30 mL) was added to benzyl bromide . The mixture was stirred at room temperature for 24 h. After filtration to remove the unreacted solid and drying under reduced pressure, the residue was purified by column chromatography on silica gel using dichloromethane as eluent to give the title compound cd]oxadiphosphinine 1,3-disulfide  and freshly dried and finely ground KF  in dry acetonitrile (50 mL) was stirred at ambient temperature. After mixing for 10 min, the mixture was heated at 80 °C for 2 h. Upon cooling to room temperature the reaction mixture was filtered and the filtrate was evaporated in vacuo to give the title compound 11 as pale yellow solid . M.p. 170–171 °C. Selected IR : 1494 (m), 1219 (m), 1159 (m), 1029 (m), 926 (vs), 847 (s), 821 (s), 762 (s), 682 (vs), 650 (vs), 584 (s), 561 (s), 438 (m). 1H-NMR , 8.53  = 8.2 Hz, J = 1.4 Hz, 1H, Ar-H), 8.48–8.44 , 8.21  = 8.2 Hz, 1H, Ar-H), 8.04–8.01 , 7.70–7.61  ppm. 13C-NMR , 140.0  = 95.5 Hz, Ar-C), 134.9 , 134.1  = 14.5 Hz, Ar-C), 131.9  = 15.6 Hz, Ar-C), 130.9 , 126.4 , 126.1 , 125.1 , 124.9 , 117.0  ppm. 31P-NMR , 107.1  = 54.0 Hz), 78.1  = 1099 Hz, 2J = 54.0 Hz) pm. 19F-NMR , −42.6  = 1104 Hz) ppm. MS , 319 [C10H7FOP2S3]−. Accurate mass measurement (CI−MS): 318.9030 [C10H7FOP2S3]−, calculated mass for [C10H7FOP2S3]−: 318.9040.A mixture of 1,3-epithionaphthooxadiphosphinine 1,3-disulfide  was added dropwise tetrabutylammonium fluoride  at room temperature. The off-white suspension disappeared immediately and a brown clear solution formed. The mixture was stirred for one more hour for completion. After filtering through a Celit lawyer the filtrate was evaporated in vacuo to give the title compound 12 as brown sticky paste . Selected IR : 1485 (s), 1381 (m), 1218 (m), 1162 (m), 947 (s), 904 (m), 852 (m), 772 (m), 695 (s), 642 (m), 587 (s), 552 (m). 1H-NMR , 8.52–7.42 , 3.13  = 8.0 Hz, 8H, CH2), 1.55–1.53 , 1.33  = 8.0 Hz, 8H, CH2), 0.92  = 8.0 Hz, 12H, CH3) ppm. 13C-NMR , 142.0  = 95.8 Hz, Ar-C), 134.8 , 134.1  = 15.6 Hz, Ar-C), 131.7  = 14.5 Hz, Ar-C), 130.4 , 128.9 , 126.7 , 126.4 , 125.2 , 124.9 , 58.7 (CH2), 23.9 (CH2), 19.7 (CH2), 13.5 (CH3) ppm. 31P-NMR , 106.9  = 54.0 Hz), 78.6  = 1099 Hz, 2J = 54.0 Hz) pm. 19F-NMR , −28.4  = 1104 Hz, 3J = 3.2 Hz) ppm. MS , 242 [C16H36N]+. Accurate mass measurement (CI+MS): 242.2841 [C16H36N]+, calculated mass for [C16H36N]+: 242.2842; MS , 319 [C10H7FOP2S3]−. Accurate mass measurement (CI−MS): 318.9032 [C10H7FOP2S3]−, calculated mass for [C10H7FOP2S3]−: 318.9040.To a suspension of 1,3-epithionaphthooxadiphosphinine-1(3H)-thiolate 1,3-disulfide (0.358 g. 1.0 mmol) was dissolved in degassed water (10 mL). To this solution was added dropwise an equivalent amount of tetraphenylphosphonium chloride  in water (10 mL). The precipitate almost immediately formed and the mixture was allowed to continue stirring for 1 h. The salt was harvested by filtration and was dried over 100 °C in vacuo to give the title compound 13 as off-white solid . M.p. 163–165 °C. Selected IR : 1585 (m), 1481 (m), 1436 (s), 1185 (m), 1163 (m), 1107 (vs), 922 (s), 826 (s), 753 (s), 72 3(s), 689 (vs), 647 (s), 583 (s), 526 (vs). 1H-NMR , 8.52–8.31 , 8.09  = 8.3 Hz, 1H, Ar-H), 7.91–7.85 , 7.75–7.71 , 7.64–7.56  ppm. 13C-NMR , 141.9  = 96.0 Hz, Ar-C), 135.7  = 3.2 Hz, Ar-C), 134.6 , 134.5 , 134.4 (Ar-C), 134.0 (Ar-C), 133.9 (Ar-C), 131.7 (Ar-C), 131.6 (Ar-C), 130.6  = 12.5 Hz, Ar-C), 130.1 (Ar-C), 126.5  = 10.8 Hz, Ar-C), 124.9  = 12.5 Hz, Ar-C), 117.9 , 117.1  ppm. 31P-NMR , 106.9  = 54.0 Hz), 78.2  = 1099 Hz, 2J = 54.0 Hz), 23.8 (s) pm. 19F-NMR , −28.7  = 1100 Hz, 3J = 3.2 Hz) ppm. MS , 339 [C24H20P]+. Accurate mass measurement (CI+MS): 339.1296 [C24H20P]+, calculated mass for [C24H20P]+: 339.1297. MS , 319 [C10H7FOP2S3]−. Accurate mass measurement (CI−MS): 318.9031 [C10H7FOP2S3]−, calculated mass for [C10H7FOP2S3]−: 318.9040.Potassium3-fluoronaphthooxadiphosphinine-1(3H)-thiolate 1,3-disulfide  was dissolved in degassed water (5 mL). To this solution was added dropwise an equivalent of 1,3,4-triphenyl-1H-1,2,4-triazol-4-ium tetrafluoroborate  in water/acetone . The mixture solution was stirred at room temperature for 2 h. Then, the organic solvent was evaporated under reduced pressure and the pale yellow precipitate was almost immediately out. After filtration and washing with water (2 × 5 mL) and ethyl ether (5 mL), the precipitate was dried at 100 °C in vacuum to give the title compound 14 as brown solid . M.p. 154–155 °C. Selected IR : 1586 (m), 1559 (s), 1485 (s), 1460 (m), 1447 (m), 1340 (m), 1239 (m), 1061 (s), 936 (s), 841 (m), 762 (s), 687 (vs), 644 (s), 584 (s). 1H-NMR , 11.20 , 8.37–8.07 , 7.66–7.39  ppm. 13C-NMR , 154.0 (triazol-C), 142.0 (triazol-C), 135.0 (Ar-C), 134.8 (Ar-C), 134.5 (Ar-C), 134.2 (Ar-C), 132.7 (Ar-C), 131.7 (Ar-C), 131.2 (Ar-C), 130.9 (Ar-C), 130.5 (Ar-C), 130.2 (Ar-C), 129.8 (Ar-C), 129.7 (Ar-C), 129.5 (Ar-C), 129.3 (Ar-C), 128.7 (Ar-C), 128.5 (Ar-C), 128.2 (Ar-C), 126.6 (Ar-C), 126.3 (Ar-C), 125.3 (Ar-C), 125.0 (Ar-C), 124.4 (Ar-C), 122.0 (Ar-C), 121.3 (Ar-C) ppm. 31P-NMR , 106.5  = 54.0 Hz), 78.6  = 1106 Hz, 2J = 54.0 Hz) pm. 19F-NMR , −28.5  = 1106 Hz) ppm. MS , 298 [C20H16N3]+. Accurate mass measurement (CI+MS): 298.1335 [C20H16N3]+, calculated mass for [C20H16N3]+: 298.1339; MS , 319 [C10H7FOP2S3]−. Accurate mass measurement (CI−MS): 318.9031 [C10H7FOP2S3]−, calculated mass for [C10H7FOP2S3]−: 318.9040.Potassium3-fluoronaphthooxadiphosphinine-1(3H)-thiolate 1,3-disulfide  was dissolved in degassed water (10 mL). To this solution was added dropwise an equivalent amount of 1,3-dimesityl-4,5-dihydro-1H-imidazol-3-ium chloride  in water (10 mL). The precipitation was almost immediately formed and the mixture was allowed to continue stirring for 2 h. The precipitate was harvested by filtration and dissolved in dichloromethane (30 mL). The solution was dried over MgSO4 and dried in vacuo to give the title compound 15 as white foam . Selected IR : 1628 (vs), 1481 (m), 1378 (m), 1261 (s), 1217 (m), 1162 (m), 942 (s), 904 (m), 849 (m), 82 5(m), 769 (m), 691 (s), 643 (s), 585 (s). 1H-NMR , 8.61 , 8.32–7.34 , 7.00 , 4.49 , 3.66 , 2.36 , 2.32 , 1.80  ppm. 13C-NMR , 159.7 (imidazol-C ), 140.9 (Ar-C), 135.1 (Ar-C), 134.6 (Ar-C), 34.2 (Ar-C), 134.0 (Ar-C), 131.7 (Ar-C), 131.5 (Ar-C), 130.3 (Ar-C), 130.1 (Ar-C), 129.6 (Ar-C), 126.5 (Ar-C), 126.3 (Ar-C), 125.1 (Ar-C), 124.9 (Ar-C), 53.1 (imidazol-C), 51.8 (imidazol-C), 20.9 (CH3), 17.9 (CH3) ppm. 31P-NMR , 106.4  = 54.0 Hz), 78.2  = 1101 Hz, 2J = 54.0 Hz) pm. 19F-NMR , −28.7  = 1100 Hz, 3J = 3.3 Hz) ppm. MS , 307 [C21H27N2]+. Accurate mass measurement (CI+MS): 307.2170 [C21H27N2]+, calculated mass for [C21H27N2]+: 307.2169; MS , 319 [C10H7FOP2S3]−. Accurate mass measurement (CI−MS): 318.9031 [C10H7FOP2S3]−, calculated mass for [C10H7FOP2S3]−: 318.9040.Potassium3-fluoronaphthooxadiphosphinine-1(3H)-thiolate 1,3-disulfide  or tetrabutylammonium ferrocenylphosphonofluoridodithioate  and dry dichloromethane (10 mL) was stirred at room temperature for 48 h. After removing the unreacted solid by filtration the remaining filtrate was concentrated to 2 mL and was purified by column (eluted by dichloromethane) to give the title compound 18 as yellow foam  for both starting materials 11 and 12. M.p. 78–80 °C. Selected IR : 1584 (m), 1483 (m), 1436 (s), 1317 (m), 1186 (m), 1159 (m), 1107 (s), 996 (m), 890 (m), 825 (m), 768 (m), 723 (s), 687 (s), 590 (s), 525 (vs), 453 (m). 1H-NMR , 7.91–7.84 , 7.76–7.69 , 7.63–7.55  ppm. 13C-NMR , 141.7  = 3.0 Hz, Ar-C), 138.6  = 10.8 Hz, Ar-C), 134.3  = 13.0 Hz, Ar-C), 130.5  = 117.9 Hz, Ar-C), 129.9 (Ar-C), 124.6 (Ar-C) ppm. 31P-NMR , 106.9  = 54.0 Hz) pm. MS , 348 [C10H6O2P2S4]+. Accurate mass measurement (EI+MS): 347.8719 [C10H6O2P2S4]+, calculated mass for [C10H6O2P2S4]+: 347.8727.A mixture of potassium3-fluoronaphtho−. Accurate mass measurement (CI−MS): 283.2954 [C12H9FOPS2]−, calculated mass for [C12H9FOPS2]−: 283.2951.A mixture of 2,4-bis(4-phenoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide (Belleau’s Reagent)  and freshly dried and finely ground KF  in dry acetonitrile (50 mL) was stirred at ambient temperature. After mixing for 10 min, the mixture was heated at 80 °C for 2 h. Upon cooling to room temperature the reaction mixture was filtered and the filtrate was evaporated 20 as slightly greenish yellow oil . Selected IR : 1595 (s), 1499 (s), 1426 (m), 1292 (m), 1252 (s), 1177 (m), 1108 (s), 1027 (m), 831 (m), 800 (m), 738 (m), 691 (s), 554 (m). 1H-NMR , 8.08–7.91 , 6.87–6.81 , 3.23–3.04 , 1.63–1.51 , 1.37 , 0.98  = 7.3 Hz, 12H, CH3) ppm. 13C-NMR , 162.2, 160.9, 136.5  = 112.0 Hz, Ar-C), 131.6  = 13.6 Hz, Ar-C), 131.4 , 113.0 , 112.8 , 112.5 , 58.8 (CH2), 24.0 (CH2), 19.7 (CH2), 13.4 (CH3) ppm. 31P-NMR , 126.3  = 1016 Hz) pm. 19F-NMR , −20.9  = 1017 Hz) ppm. MS , 242 [C16H36N]+. Accurate mass measurement (CI+MS): 242.2841 [C16H36N]+, calculated mass for [C16H36N]+: 242.2842; MS , 283 [C12H9FOPS2]−. Accurate mass measurement (CI−MS): 283.2953 [C12H9FOPS2]−, calculated mass for [C12H9FOPS2]−: 283.2951.To a suspension of 2,4-bis(4-phenoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide (Belleau’s Reagent)  in THF (30 mL) was added dropwise tetrabutylammonium fluoride  at room temperature. The off-white suspension disappeared immediately and a colorless solution formed. The mixture was stirred for one more hour for completion. After filtering through a Celit lawyer, the filtrate was evaporated in vacuum to give the title compound 4 to give the title compound 21 as white form  after removing the solvent and drying in vacuo. Selected IR : 1594 (m), 1497 (m), 1438 (m), 1250 (m), 1179 (m), 1108 (s), 1024 (m), 830 (m), 800 (m), 723 (s), 688 (s), 554 (m), 526 (s). 1H-NMR , 8.04–7.87 , 7.77–7.73 , 7.65–7.57 , 6.79–6.76  ppm. 13C-NMR , 160.7, 135.8  = 3.1 Hz, Ar-C), 134.6 (Ar-C), 134.5 (Ar-C), 132.7 (Ar-C), 132.6 (Ar-C), 131.3  = 14.5 Hz, Ar-C), 130.7  = 13.5 Hz, Ar-C), 119.3 (Ar-C), 116.9 (Ar-C), 112.6 (Ar-C), 112.3 (Ar-C) ppm. 31P-NMR , 126.4  = 1019 Hz), 23.8 (s) pm. 19F-NMR , −35.3  = 1018 Hz) ppm. MS , 339 [C24H20P]+. Accurate mass measurement (CI+MS): 339.1297 [C24H20P]+, calculated mass for [C24H20P]+: 339.1297; MS , 283 [C12H9FOPS2]−. Accurate mass measurement (CI−MS): 283.2952 [C12H9FOPS2]−, calculated mass for [C12H9FOPS2]−: 283.2951.Potassium (4-phenoxyphenyl)phosphonofluoridodithioate (0.322 g. 1.0 mmol) was dissolved in degassed water/acetone . To this solution was added dropwise an equivalent amount of tetraphenylphosphonium chloride  in water (10 mL). The mixture was allowed to continue stirring at room temperature for 3 h. After removing the organic solvent, the mixture was extracted with dichloromethane (2 × 30 mL). The organic lawyer was washed with water and dried over MgSO2, FcLR} with dry KF or tetrabutylammonium fluoride, led to the corresponding potassium and tetrabutylammonium salts of phenyldithiofluorophosphinic acids in excellent yields. Potassium phenyldithiofluorophosphinic acid is readily converted into the corresponding organic adduct by treating it with an equimolar amount of tetraphenylphosphonium chloride, and treating potassium phenyldiselenofluorophosphinic acid with mono- and di-halogenated alkanes gave rise of a series of the corresponding esters of phenylphosphonofluoridodithioates in high yields. Furthermore, using 1,3-epithionaphthooxadiphosphinine 1,3-disulfide or Belleau’s reagent in its place resulted in the formation of a series of novel salts, adducts, and ester derivatives.In summary, the reaction of the ferrocene analogue of Lawesson reagent, 2,4-diferrocenyl-1,3,2,4-diathiadiphosphetane 2,4-disulfide {[FcP(μ-S)S]


The large number of South Africans with disability who cannot access good quality rehabilitation presents a public health and human rights challenge. A cost-effective, efficient approach is required to address this. Implementation of high-quality, contextually relevant clinical practice guidelines (CPGs) could be a solution; however, this requires significant investment in innovative capacity-building.A qualitative descriptive national study was conducted to explore the perspectives of South African stakeholders in rehabilitation, regarding CPG capacity-building. Twenty rehabilitation professionals  were interviewed. Transcribed interview data were analysed using a deductive content analysis approach, mapping findings to an international capacity-building framework to produce new knowledge.Capacity-building is required in content, purpose and construction of locally relevant CPGs, as well as personal, workforce and systems capacity. Principles and strategies were derived to underpin implementation of CPGs that are user friendly, context specific, relevant to the needs of end-users, and achievable within available resources. Collaboration, networks and communication are required at national, provincial and regional level, within and between sectors. A central agency for CPG methods, writing, implementation and evaluation is indicated.South African rehabilitation can benefit from a multi-level CPG capacity-building focusing on performance, personal, workforce and systems issues. Rehabilitation based on best-available evidence has the potential to cost-effectively optimise health outcomes, quality of life and well-being . It is eWHO recognises the need to reform healthcare, and is advocating for rehabilitation to be included into universal health coverage , 3. SoutEffective production and implementation of CPGs relates to local capacity , 3. Capacapacity challenge” and “dimensions of issues and needs” whilst describing the goal of capacity-building as “to tackle problems that are related to policy and methods of development, whilst considering the potential limits and needs of the people of the country concerned”  Project  , Stellenbosch University (South Africa) (N14/02/ 008) and University of South Australia, Australia (0000034923).The six-person research team comprised four individuals with experience in CPG writing and implementation, who also had backgrounds in rehabilitation , a public health epidemiologist (SM) with expertise in systems analysis, and a social scientist (HP) with expertise in qualitative methods and analysis. All team members were experienced in conducting and reporting qualitative research.At the time the research was conducted, there was no published information about primary care AH or rehabilitation CPG activity in South Africa, and no formal networks to support robust sampling , 11, 15.We used a cluster sampling framework that supported effective, efficient and comprehensive maximum variation sampling , 16, 17.We invited nominated individuals to participate in individual semi-structured interviews. The interview schedule allowed exploration of a broad range of issues related to CPG need, writing and implementation in primary healthcare settings in South Africa. Interview questions sought to obtain information on (1) existing frameworks, supports and activities that are used to develop and implement CPGs in rehabilitation, (2) current roles, skills and availability of resources available to support implementation of CPGs and what additional resources are required, (3) barriers and facilitators for the development and implementation of CPGs, and (4) the contexts in which rehabilitation CPGs are formulated and implemented. The interviewer encouraged exploration of responses using a combination of conventional interview techniques  . InterviParticipants provided formal written consent for audio recordings. Where participants were not comfortable providing formal consent for recording the entire interview, notes were taken during periods where information was provided ‘off the record’. This assisted the researchers to validate and contextualise the formally consented material without breaching confidence.Researchers conducted interviews in pairs, which provided consistency and coherence in data collection, handling and analysis , 17. RegOnce we suspected that we had reached saturation in any cluster (where nothing new was heard since the last interview), one further interview was conducted with the next participant. If no new information was forthcoming, interviewing in that cluster (not discipline) stopped at that point. However, if new information was found in that interview, further sampling and interviewing occurred until data saturation was achieved.One researcher (QL) was known to eight participants, and a second researcher (KG) was also known to four of these. When inviting participants to join the research, the researchers declared their position (and prior knowledge), and the intent of the research. They clarified ways in which participant anonymity would be protected. Prior to the interview, all interviewees reviewed the information sheet, signed the consent form, and clarified issues and concerns.We applied an explorative, descriptive methodology to data analysis , 17, undaides-de-memoir. To establish reliability in theme identification, four researchers independently hand-analysed two randomly selected interviews  and discussed their findings, particularly the differences in interpretation. They then independently analysed one further interview (N18) to test agreement. During this process, the researchers started defining the key themes according to Potter’s framework  outcomes or the physio or the medical or whatever, so it’s a whole team approach. Then maybe also giving links to other resources that the new graduates could consult for extra input if they needed it.” “Participants in policy and management positions noted that, in South Africa, there was limited capacity to develop CPGs across all health areas, not just in rehabilitation. To counter this, guidance for priority programmes such as the HIV and TB had been adapted from CPGs developed by international institutions. Participants who are aware of this practice indicated the importance of contextualising CPGs to suit the national and provincial contexts. South Africa is a country with marked inequality and disease profiles that differ between geographical regions . The folSome of the guidelines that we use are approved guidelines that come from the World Health Organization and then we adapt them through the national directorate and adapt them onto our province and use them and where we are having challenges are been reported back to national and provide feedback, we are not able to provide this with limited resources and when we need further training then we ask experts sometimes to be provided by the national department to come and assist in conducting training on how to implement guidelines that are coming from them.” “This was mentioned many times as an essential step to help individuals to understand, write and implement CPGs. Personal empowerment was seen as essential to enhance individual and organisational uptake of South Africa-specific rehabilitation CPGs. Participants indicated that all rehabilitation stakeholders should be equipped with the knowledge, skills, willingness and confidence to at least use CPGs, if not to participate in writing them. However, from the interviews, it was clear that even basic understanding of what CPGs were was lacking. For instance, virtually all participants asked clarifying questions to determine if the interview was related to policy documents, protocols or guidelines. A quote by this rehabilitation manager highlights this issue:Very few guidelines available at the moment. What exactly do you mean by a guideline?” “Personal training was viewed as an essential strategy to address individual knowledge and skill gaps. Some participants gave suggestions about training content such as providing basic information about guideline taxonomies. There was also a strong sense that training should also encompass implementation and subsequent monitoring. Continual professional development opportunities were suggested as a feasible pathway for training as this quote suggests.I guess, I mean national associations are one, continuing professional development opportunities is the obvious one as facilitators of guideline implementation, …” “Participants also noted that an important prerequisite to building capacity is the ongoing availability of experienced trainers. Therefore, the development of future trainers was suggested as an important aspect of building CPG capacity. Contingency planning is a particular challenge, considering the high staff turn-over rates within the public healthcare system. Building capacity not only in training but also of trainers to advocate for CPG use was viewed as a crucial element for sustainability.In terms of going forward I am hoping that we will retain because people have developed quite a bit of skill in specifically in their specific areas around adaptation or contextualisation of guidelines, ...” “So we brief them, we train them, we give them information and we all in agreement then they as a team in their own districts, they will cascade, all we do is support and monitor for those who do not have capacity to train or give out the information.” “A common theme was the significant shortage of rehabilitation workers in South Africa. A rehabilitation manager responsible for planning rehabilitation services indicated that staff shortages are critical to address when building capacity in rehabilitation CPGs. To address insufficient workforce capacity, a multi-level workforce approach was suggested . ThiOver the last year or so we’ve tried to change that, so you will see now in the primary healthcare platform more and more allied health practitioners have been employed at the sub-district level, we are now piloting these mid-level workers for rehabilitation care workers and now have a technical working group that is looking at this thing.” “The consequences of workforce shortages manifest at two levels.Having dedicated time was noted by many as essential to ensure effective individual uptake of CPGs. It was also acknowledged that putting CPGs into place may reduce workplace pressures and improve patient flow, because care may be more effective and efficient. However, tensions were noted between finding time to locate and read CPGs, and to implement change, whilst dealing with the continual pressures of high patient volume and high patient to staff ratios.Time was also required when influencing and transferring skills to colleagues at different levels of care. Making changes within departments, and within organisations, required time as well as trained personnel. The quotes below from rehabilitation managers illustrate these issues.Barriers [for guideline implementation] would be, which is one I hate to admit, lack of staff. You know, you just don’t have the staff available.” “Staff turnover, new people and the people that you train they usually don’t cascade the information as well because they won’t be having time.” “Implications for CPG implementation are that efforts should be rewarded, and the interventions sustainable. This can refer to workforce, consumables, financial resources, effort and goodwill, and behaviour changes. As this rehabilitation director noted:The support that is needed in terms of associates to implement whatever guideline. If you are having a clinical guideline the assumption is that you have the resources to implement. If you need consumables the assumption is that consumables must be there but we don’t always have all the consumables needed.” “Participants indicated that a poor understanding of rehabilitation by other healthcare providers may be a factor influencing the uptake of CPGs at primary care level. This may well be related to overt historical focus on mortality from communicable diseases in South Africa  and the The doctors, our managers, the rest of the team needs to buy in to this to understand what we’re doing. They need to obviously, I would love for them to contribute to what we’re doing.” “Seamless integration of rehabilitation into all levels of care (from primary to quaternary) must be addressed, as participants noted that rehabilitation services seem to be biased towards the higher levels of care. Primary healthcare is seen as an inexpensive, client-focused care level which, if better resourced, could deal with many more patients, more effectively. The current fragmented rehabilitation service delivery approach is not efficient or effective, as one rehabilitation director suggested.Healthcare 2010 and Healthcare 2030 we’ve always identified rehabilitation and not just in the Western Cape, nationally as well but we have never ever put our money where our mouth is and as a result the rehabilitation services are fragmented, they are largely in the metro and in the tertiary hospitals.” “The importance of communication was highlighted, between different levels of care, provinces, team members, professional associations, and educational and government institutions. A physiotherapist involved in CPG development indicated that CPG implementation could be facilitated via good dialogue with professional societies :So the support that we had from the Society through the consultant really helped because I mean if I had to be the only person kind of driving it.” “In South Africa, all newly qualified therapists are required to do 1 year of community service. These community placements are usually within rural and remote areas, and the new graduates are often sole practitioners. CPGs would assist them in delivering evidence-based rehabilitation, particularly if they were supported by good communication with mentors and were involved in supportive collaborative partnerships. The speech and language therapist below suggests that CPGs can also play a role in supporting new graduates who are often working independently in remote regions, and with little guidance.So firstly, it’s equipping them with knowledge and skill to deal with that. So that’s one. But actually, there’s very little support provided for people in com-serve [community service] for that kind of thing.” “The need to work collaboratively with government departments, educational institutions, and healthcare managers and clinicians was regularly expressed. Leveraging off combined resources and diverse skill sets, the development and implementation of good quality CPGs can be facilitated in a way that met different professional agendas. However, concern was expressed about the lack of productive partnerships due to issues such as tensions between clinicians and academics.We struggle to find people that can sort of lead the profession in development of guidelines and implementation of guidelines, because it’s like the clinicians on one side and the academics on the other side.” “Lack of financial resources for CPG writing, training and implementing was perceived to be a major barrier for CPG use in rehabilitation. Some of the participants suggested that guaranteed central funding streams were required so that CPG activity could be planned and have tangible, achievable timelines.Look, there are a number of ways, one where what we are able to do, nationally, we can bring together people to be together in the way of training the trainer but for more localised training some province might be able to have their own provincial and fund it but what the, the likely to be able to fund would be a facilitation fee and then institutions, institutions pay for travel, accommodation but the province is able to pay for facilitation and the venue.” “Table Developed from this mapping process was a set of principles and strategies specific to South African rehabilitation capacity-building to better use contextually relevant rehabilitation CPGs. These principles and strategies could underpin discussions about the delivery of cost-effective, equitable and efficient rehabilitation, improve the lives of individuals with disability, and enable provincial and national governments to meet chronic healthcare targets. The principles and strategies are outlined in Table This is the first paper, as far as we are aware, that explores capacity development in rehabilitation CPGs from the perspectives of national stakeholders in a country with huge disability burden rehabilitation. The application of a deductive methodology building on an established capacity-building framework  enabled The strengths of the study were its nationally representative sample of key informants with views to share about capacity-building in rehabilitation in South Africa. We attempted to develop a sampling framework that allowed us to capture as many representative rehabilitation stakeholder voices as possible in the complex environment that is South African primary healthcare. However, we may not have captured all relevant perspectives. We also applied a structured deductive framework to assist us to make sense of the rich interview data, and to produce summary information that could guide capacity-building around South African rehabilitation CPGs. We chose an internationally tested framework that appeared to resonate with our context, aims and data . DespiteThe design, practicality and local relevance of rehabilitation CPGs were highlighted as essential elements to improve CPG uptake in primary care. This concurs with published reports that state that barriers to CPG use are the length of guidelines, the lack of relevant strategies and facilitation for implementation and environment, and organisational constraints . These fContextualisation of CPGs requires methodological knowledge and skills that are not as well understood as de novo CPG development skills –27. HoweAt systems level in South Africa, a major implementation challenge will be to advocate for better integration of rehabilitation into primary care systems. The South African National Department of Health is redesigning its approach towards an integrated healthcare system  , 24. OurCapacity development linked to collaboration with higher education institutions is a potentially useful strategy to facilitate the uptake of CPGs. Such collaboration is also important for supporting local research in the fields of CPG contextualisation and new writing methods. Higher education institutions can become involved in CPG training as a potential avenue to upskill local rehabilitation professions and meet enrolment and income generation targets. These CPD activities should be affordable, particularly to therapists working at primary care level with the public sector. CPD activities should be well coordinated to prevent fragmentation and resource wastage, which will hamper progress in building CPG capacity. Training programmes should be comprehensive and include strategies to implement, monitor and evaluate the use of CPGs. Such national data will be essential to gain support from health policy-makers.Understanding, accessing and implementing rehabilitation CPGs in South Africa will continue to be a challenge for all health professionals, unless the suggested principles and strategies can be actioned , 24, 25.Capacity-building in CPG implementation and uptake is crucial in rehabilitation, particularly in the South African context. South African rehabilitation services can benefit from a multi-level CPG capacity-building approach focusing on the performance, personal, workforce and systems levels. However, challenges in building capacity in CPG uptake at the South African primary care level are not unique to rehabilitation. Improved quality care using CPGs could be well addressed by a national steering body, tasked with initiating and managing a CPG repository in South Africa.


Drosophila fat body is the primary organ of energy storage as well as being responsible for the humoral response to infection. Its physiological function is of critical importance to the survival of the organism; however, many molecular regulators of its function remain ill-defined. Here, we show that the Drosophila melanogaster bromodomain-containing protein FS(1)H is required in the fat body for normal lifespan as well as metabolic and immune homeostasis. Flies lacking fat body fs(1)h exhibit short lifespan, increased expression of immune target genes, an inability to metabolize triglyceride, and low basal AKT activity, mostly resulting from systemic defects in insulin signalling. Removal of a single copy of the AKT-responsive transcription factor foxo normalises lifespan, metabolic function, uninduced immune gene expression and AKT activity. We suggest that the promotion of systemic insulin signalling activity is a key in vivo function of fat body fs(1)h.The This article has an associated First Person interview with the first author of the paper. Summary: The bromodomain-containing protein FS(1)H is required in the Drosophila fat body for normal lifespan and metabolic and immune function, largely via the insulin pathway. Drosophila fat body is the primary site of energy storage as well as being the source of the transcriptionally induced humoral immune response   . AMP tras (AMPs) . The Tol factors . AMPs ar factors .Drosophila, there are eight insulin-like peptides (ILPs), of which three – ILP2, ILP3 and ILP5 – are believed to play roles analogous to that played by insulin in regulating physiology in response to transient changes in nutritional state  homeotic (fs(1)h) encodes the sole Drosophila member of the bromodomain and extra-terminal motif (BET) protein family, homologous to mammalian BCPs BRD2/3/4/T (in vivo roles of BET domain proteins in immune regulation are not entirely clear.Bromodomain-containing proteins (BCPs) are well characterised as regulators of transcription. The bromodomain, originally described as a domain common to residues . In tranresidues . In partresidues . female D2/3/4/T . These pD2/3/4/T . In receD2/3/4/T . Howeverfs(1)h, the sole Drosophila BET-family protein, is required in the fat body for normal lifespan and immune and metabolic function. Flies with fat-body-specific fs(1)h knockdown exhibit abnormally high expression of AMPs, whether uninfected or infected with bacteria; low levels of free sugar, combined with an inability to utilise triglyceride; and an extremely short lifespan. fs(1)h knockdown animals also exhibit significantly reduced systemic AKT activity, which is the underlying cause of nearly all these observed phenotypes. This effect may result from increased expression of several secreted ILP antagonists. Removal of a single copy of the AKT-responsive transcription factor foxo ameliorates almost all of the observed phenotypes, restores the lifespan of these animals to normalcy and normalises expression of insulin-regulatory signals, suggesting that much of the in vivo activity of fs(1)h in this tissue is mediated via foxo regulation.We show here for the first time that fs(1)h knocked down in the fat body exhibited dramatically reduced survival even when uninfected . This phenotype was equally strong with a second, non-overlapping fs(1)h dsRNA  and with r4-Gal4, a second fat-body driver  (fs(1)h knockdown flies exhibited gut barrier dysfunction than the control genotype, although not necessarily to the levels that have been reported for wild-type 1118w flies dying of old age  . Gut barig. S1C) . In keep old age B.Fig. 1Drosophila (fs(1)h knockdown flies by testing expression of AMPs. We observed significant increases in AMP expression in fs(1)h knockdown animals . This effect was also present in uninfected flies as well as in flies that had been infected with bacteria, and was observed for most AMPs tested, including targets of both Toll and IMD pathways . This indicates that fs(1)h is an endogenous negative regulator of AMP expression, acting in the presence and absence of infection.The fat body is the primary site of the humoral immune response in osophila . AMPs arosophila . Therefofs(1)h knockdowns, we assayed REL protein expression and processing . We found that total REL abundance was dramatically increased in fs(1)h knockdowns, but the quantity of the C-terminal cleavage product, indicative of pathway activation, was not different between wild-type controls and fs(1)h knockdowns; this was true whether the flies were infected or uninfected. This suggests that the increase in baseline AMP expression does not reflect an overall increase in IMD pathway activity.To determine whether known immune signalling pathways were involved in the increased AMP expression observed in ocessing ; REL is fs(1)h knockdown flies.In addition to its role in immunity, the fat body is the primary location of triglyceride and glycogen storage and a key gluconeogenic tissue. We next assayed metabolic stores and free sugar levels in fs(1)h knockdowns was heterogeneous; we sometimes observed essentially normal triglyceride levels, while at other times triglycerides were elevated roughly twofold. However, we consistently observed that, when starved, fs(1)h knockdowns were unable to utilize their triglyceride stores (fs(1)h knockdown also affected carbohydrate metabolism: in these animals, we observed reductions in glycogen (stored carbohydrate) as well as in free glucose and trehalose (circulating sugars) h sugars) G. These  glucose .brummer (bmm) and Hormone sensitive lipase (Hsl) and the perilipins Plin1 and Plin2. Of these genes, the perilipins and bmm were reduced in expression, although not eliminated h knockdowns. AKT is the key effector kinase of the insulin signalling pathway; its activity is stimulated by phosphorylation at serine 505 (Ser505). We found a significant reduction in levels of phospho-Ser505 AKT in fs(1)h knockdowns relative to controls (fs(1)h knockdown, and was also visible  with r4-Gal4, a second fat-body driver .The insulin signalling pathway has conserved roles in the regulation of metabolism and lifespan, and can also affect AMP expression . Based ocontrols A. Again,fs(1)h knockdowns were perfectly capable of activating AKT in response to exogenous insulin . We tested our flies for insulin resistance by testing their response to injected human insulin. We found that  insulin B. This ifs(1)h knockdown was not directly inhibiting ILP expression  (fs(1)h knockdowns  and found either small changes, or elevated expression, indicating that pression  Fig. 2Cig. S2C) . To resoig. S2C) . We obseCig. S2C conv, Sdr and ImpL2, as well as CG10702, which encodes an apparent insulin receptor homologue lacking the kinase domain and thus might also function as an ILP antagonist (fs(1)h knockdowns (fs(1)h knocked down by the GD51227 line .These observations together indicated that the observed defect in AKT activity might be caused by increased expression of one or more extracellular antagonists of ILP signalling. We assayed the expression of the characterised ILP antagonists tagonist . All fouockdowns E, suggesfs(1)h knockdown flies, we combined fs(1)h knockdown with a loss-of-function mutation in foxo. Remarkably, we observed that heterozygosity for a foxo-null allele was sufficient to completely rescue the lifespan defect of fs(1)h knockdown flies h knockdown, but foxo heterozygosity had no effect on Gal4 expression .The best-characterised transcriptional effector downstream of AKT is FOXO. FOXO is phosphorylated by AKT, promoting its nuclear exclusion, so that reducing AKT activity should increase FOXO activity. To determine the extent to which FOXO hyperactivation was responsible for the phenotypes of wn flies A. This cfoxo heterozygosity on AMP expression in fs(1)h knockdowns. We found that AMP expression was almost entirely normalised in uninfected fs(1)h-knockdown animals also lacking one copy of foxo . Together, these data indicate that fs(1)h affects baseline and infection-induced AMP expression via distinct mechanisms: baseline AMP overexpression in fs(1)h knockdowns likely results from FOXO hyperactivation, while infection-induced AMP overexpression results from some other mechanism, possibly the observed overexpression of REL.We next tested the effect of  of foxo B, in acc of foxo . Howeverfoxo and fs(1)h on metabolic phenotypes. foxo heterozygosity increased total triglyceride levels on an otherwise wild-type background; this phenotype was unaltered by fs(1)h knockdown, but fs(1)h knockdown animals lacking one copy of foxo regained their ability to utilise stored triglyceride (fs(1)h knockdowns also lacking one copy of foxo, while glycogen levels were independent of foxo genotype (fs(1)h knockdowns were markedly short-lived when starved; this effect was also ameliorated by foxo heterozygosity, in keeping with the improved ability of these animals to utilise stored triglyceride .We then examined the combined effect of lyceride D. Levelsgenotype E. Finallfoxo suggested that fs(1)h knockdown could also cause insulin-pathway loss-of-function phenotypes during development that might be visible as a change in adult size. We tested this by measuring wing size and total dry mass in fs(1)h knockdowns. We found that animals with fs(1)h knocked down in the fat body exhibited a non-significant trend toward reduced dry mass, accompanied by a small increase in wing size . These data suggest that the effect of fat-body fs(1)h knockdown on systemic insulin activity is not sufficient to strongly alter larval growth.The strong phenotypic rescue we observed in adult flies heterozygous for foxo and fs(1)h, we tested the effect of foxo heterozygosity on AKT activation in fs(1)h knockdowns. We found that foxo heterozygosity was sufficient to rescue systemic AKT phosphorylation to normal levels . REL protein levels were also normalised by foxo heterozygosity h on one another's expression. We found that fs(1)h knockdown reduced foxo transcript levels significantly, an effect that was unaltered by compound foxo heterozygosity h expression, foxo-null mutants expressed fs(1)h at significantly higher levels than wild-type controls (We next examined the effects of zygosity C. In turcontrols D. Togethfs(1)h loss-of-function in the fat body are primarily mediated via effects on systemic regulation of physiological signalling, and that these effects are largely dependent on foxo.Overall, our data indicate that the biological consequences of fs(1)h, encoding the sole Drosophila member of the BET bromodomain protein family, has critical functions in both immunity and metabolism: it is required for normal utilisation of triglyceride stores, to restrain expression of AMPs and to enable normal survival. Remarkably, it appears that almost all of these functions are due to effects on signalling via AKT and FOXO. fs(1)h knockdowns exhibited significant reductions in baseline AKT phosphorylation. Removal of a single copy of foxo completely rescued the short lifespan of fs(1)h knockdown animals, restored their ability to utilise triglyceride stores, and normalised their survival during starvation and their expression of AMPs when uninfected. Importantly, AMP expression was still significantly enhanced in infected fs(1)h knockdown foxo heterozygotes relative to controls, indicating that not all fs(1)h phenotypes can be attributed to foxo hyperactivation.Here, we have shown for the first time that fs(1)h knockdown is surprising, given what is known about insulin-AKT signalling functions in vertebrates and wild-type flies. Normally, we would expect impaired AKT activity to primarily impair anabolic processes; the effects we see on catabolism are unexpected. We suggest that the actual observed phenotype is a complex combination of developmental and physiological effects – flies with lifelong loss of insulin signalling exhibit increased adiposity (fs(1)h knockdown on AKT phosphorylation, and the ability of foxo mutation to completely rescue most phenotypes, suggest that fs(1)h may play an important and direct physiological role as a regulator of signalling via AKT.The overall physiological phenotype of diposity , while tdiposity . Nonethefs(1)h knockdown and the interaction with foxo appear more straightforward. foxo is able to activate AMPs directly, but it is currently not believed to be an important regulator of AMPs in the fat body during infection h is normally suppressive on AMP loci no matter what the relevant activating cue. Reduction of fs(1)h permits AMP expression, which is promoted by foxo activity in the absence of infection; AMP expression is still stronger after infection, when the relevant activating signal is provided by NF-κB activity. Further work will be required to determine whether FS(1)H is a true nodal point between immune and metabolic signalling, or is a more general regulator of gene expression that happens to affect both processes.The immune effects of nfection . We and gnalling . Conversgnalling , 2018. Wgnalling . Infectignalling . Togethein vivo may have significant metabolic consequences via effects on AKT-FOXO signalling, as well as suggesting that AKT may be a key pharmacological target of BET inhibition in anti-tumour therapy. Potential metabolic roles of Brd3 and BrdT have not been reported. However, mice lacking Brd2 exhibit some metabolic similarities with fs(1)h knockdown flies, while BRD4 inhibits autophagy in cultured human cells and in the mouse intestine :1118w;1118w; c564-Gal4;1118w; r4-Gal4 / TM6c, 1Sb;w; UAS-fs(1)h-IR (VDRC KK108662) (fs(1)h knockdown line I);w; UAS-fs(1)h-IR (VDRC GD51227) (fs(1)h knockdown line II);w; UAS-bcd-IR (VDRC KK104160) (bcd knockdown line); andw; Δ94foxo/TM6c, 1Sb .Listeria monocytogenes EGD-E and Francisella novicida U112 were used for infections. Broth cultures were grown overnight at 37°C, either still (L. monocytogenes) or shaking (F. novicida). The following day, the cultures were centrifuged at 4000 g for 10 min. The bacterial pellets were resuspended in sterile PBS and the optical density was measured at 600 nm (OD600). Listeria monocytogenes and F. novicida were injected at OD600=0.1. Infections were then performed using a pulled glass needle as previously described  at a concentration of 2.5% (wt/vol). Flies were maintained at 29°C on the dyed food for a 2-h time period in the morning. Flies were counted as a Smurf when the dye colouration could be observed outside of the digestive tract.α-Tubulin, and then analysed further. The graphed values are these tubulin-normalised abundance measurements. The qRT-PCR primers used in this study are shown in Table S1.RNA was extracted from groups of two to three flies using TRI reagent (Sigma) following the manufacturer's instructions. cDNA synthesis was carried using RevertAid Reverse Transcriptase and primed with random hexamers (Thermo Scientific). PCR was carried out with Sensimix SYBR Green no-ROX (Bioline) on a Corbett Rotor-Gene 6000. For all experiments, the cycling conditions used were as follows: hold 95°C for 10 min, then 40 cycles of 95°C for 15 s, 57°C for 30 s, 72°C for 30 s. Gene abundances were calculated based on experimental measurements on a standard curve; standards were run in every experiment, both to allow quantification and to verify that all measured values fell within the linear amplification range of the primer set. All measured mRNA abundances were first normalised to the housekeeping gene, Groups of ten flies were homogenised in 100 μl chloroform:methanol . A set of standards were prepared using lard (Sainsbury's Supermarkets Ltd) to generate a standard curve. Standards (2 μl per standard) and samples (20 μl per sample) were then loaded onto a TLC plate and migrated in a 4:1 mix of hexane:diethyl ether (both Sigma-Aldrich). TLC plates were stained using the general oxidising stain, ammonium heptamolybdate, left to dry and baked at 80°C for approximately 20 min. The plate was imaged using a Bio-Rad Molecular Imager and signal was quantitated using Bio-Rad Image Lab software.Flies were fixed for 1 h 30 min in 4% paraformaldehyde (PFA) and 0.1% Triton X-100. Samples were then washed with 1× PBS and 0.1% Triton X-100. DAPI  was added and incubated for 10 min at room temperature. The samples were washed with 1× PBS, and incubated with HCS LipidTOX Deep Red  in 1× PBS for 2 h at room temperature in the dark. Finally, the samples were mounted on glass coverslips in Fluoromount-G (eBioSciences). Images were acquired using a Leica SP5 microscope.fs(1)h-IR, n=8; c564>0, n=5) were dissected from adult male flies starved for 1 h into 1× PBS. Dissected brains were fixed in 4% formaldehyde (methanol-free) 0.1% Triton X-100 PBS (PBST) for 30 min at room temperature. Samples were then rinsed twice with 0.1% PBST and washed four times for 1 h. 5% normal goat serum in 0.1% PBST was used as a blocking agent for 1 h at room temperature. Brains were then incubated overnight at 4°C with primary antibodies in 5% normal goat serum in 0.1% PBST. The primary antibodies used were: anti-rabbit-dILP2  and anti-Rat-Elav . Brains were rinsed twice with PBST, and washed four times with 0.1% PBST for 1 h. Secondary antibodies were diluted in 5% normal goat serum in 0.1% PBST and incubated with the brains for 1 h at room temperature. The secondary antibodies used were: anti-rabbit-Alexa-Fluor-488  and anti-rat-Alexa-Fluor-555 . Samples were then rinsed twice with 0.1% PBST, and washed four times for 1 h. Brains were mounted on glass coverslips in Vectashield (Vector Laboratories). All incubations and washes were performed in a rotator. Images were acquired using a Zeiss LSM 510 confocal microscope and edited using Fiji/ImageJ.Brains ; the assay reagent was supplemented with amyloglucosidase to measure glycogen or trehalase to measure trehalose.Western blotting was carried out as previously described . PrimaryFlies were fasted for 1 h on 1% agar, then injected with 50 nl of sterile PBS (vehicle) or human insulin in PBS at a low dose (1.28 μg/ml) or a high dose (6.4 μg/ml). At 10 min post-injection, flies were homogenised in groups of three in 75 μl of 2× Laemmli loading buffer; these lysates were then assayed for AKT activity by western blot.t-tests or two-way ANOVA were used to calculate statistical significance for all experiments. For survival experiments, the log-rank and Wilcoxon tests were carried out in GraphPad Prism.For qRT-PCR, TLC and the glucose/trehalose/glycogen assay, standards were used to calculate values for each sample. The qRT-PCR analysis was also relative to α-tubulin as housekeeping control, and western blot analysis was relative to the α-tubulin loading control. Western blot and TLC images were exposed and the band densities were analysed using ImageJ software. Unpaired Fly wings were mounted on glass slides using glycerol and photographed. Areas were calculated using ImageJ. Ten wings were measured per genotype.A total of 80 flies were collected per genotype, killed by freezing, desiccated using silica gel in a closed container at room temperature for 1 week, then weighed.