Crataegus azarolus var. eu- azarolus Maire leaves, and to evaluate the therapeutic potentials particularly antimicrobial, antihyperglycemic and antihyperlipidemic of the extract and the isolated compound (3β-O-acetyl ursolic acid).Hyperglycemia is a complicated condition accompanied with high incidence of infection and dyslipidemia. This study aimed to explore the phyto-constituents of Total phenolics and flavonoidal contents were measured by RP-HPLC analysis. Free radicals scavenging activity of different extraction solvents was tested in-vitro on DPPH free radicals. The antimicrobial activity of the ethanolic extract and its fractions as well as the isolated compounds were evaluated in-vitro on variable microorganisms. Animal models were used to evaluate the antihyperglycemic and antihyperlipidemic activities of the ethanolic extract along with the isolated compound (3β-O acetyl ursolic acid).n-butanol fractions namely: ursolic acid, 3β-O-acetyl ursolic acid, ellagic acid, quercetin 3-O-β methyl ether, rutin and apigenin7-O-rutinoside. Ethanolic extract showed the highest DPPH radical scavenger activity compared to other solvents. Ethanolic extract, hexane fraction, ursolic acid, 3β-O acetyl ursolic acid and quercetin 3-O-methyl ether showed variable antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and C. albicans. Administration of the ethanolic extract or 3β-O acetyl ursolic acid orally to the mice reduced blood glucose significantly in a time- and dose-dependent manner. Ethanolic extract significantly reduced LDL-C, VLDL-C, TC and TG and increased HDL-C in rats. Ethanolic extract and 3β-O acetyl ursolic acid reduced in-vitro activity of pancreatic lipase.RP- HPLC analysis of the phenolics revealed high content of rutin, salicylic and ellagic acids. Six compounds belonging to triterpenes and phenolics were isolated from chloroform and Crataegus azarolus var. eu- azarolus Maire has the efficiency to control hyperglycemia with its associated complications. This study is the first to evaluate antihyperglycemic and antihyperlipidemic potentials of 3β-O acetyl ursolic acid.This study reveals that The online version of this article (doi:10.1186/s12906-017-1729-9) contains supplementary material, which is available to authorized users. Dyslipidemia and increased susceptibility to infections are two typical complications of diabetes mellitus. High glucose levels are highly associated with immune system impairment, particularly on neutrophils . HyperglThere has been a noticeable increase in the use of both traditional home remedies and herbal medicine instead of relying on conventional treatments . This haCrataegus azarolus is indigenous to the Mediterranean Basin. Crataegus azarolus var. eu- azarolus Maire is a low, dense, spiny tree with a beautiful inflorescence up to 6 m tall and with orange fruits /A0 ×100%.% inhibition = [A0: control absorbance, A1: absorbance of the sample, A2: absorbance of the sample in ethanol without DPPH.AStaphylococcus aureus RMTCC 3161), two representatives of the Gram-negative group (Escherichia coli RMTCC 2682 and Pseudomonas aeruginosa RMTCC 1687) and one fungus . Microorganisms were grown on appropriate media: nutrient agar for S. aureus and P. aeruginosa, MacConkey agar for E. coli and Sabouraud dextrose agar for C. albicans.Three bacterial strains and one fungus, were kindly provided by Rashid hospital (Dubai-UAE) and were used for the antimicrobial screening. This included one representative of the Gram-positive group  were used for acute toxicity and antihyperglycemic studies. Antihyperlipidemic experiments were performed on Sprague Dawley rats weighing 210 ± 5 g. All animals were kept under standard conditions, fed with regular diet and water supplied ad libitum. Mice were accommodated for 1 week prior to the experiments. All animal investigations were accepted from the Ethical Research Committee of the Dubai Pharmacy College, Dubai UAE and done according to the ethical standards of laboratory animals .50 was determined by probit test [LDbit test . Mice webit test .One night before the induction of hyperglycemia, the animals were kept fasted but given water ad libitum. The next morning, animals were injected 150 mg/kg alloxan monohydrate solution in acetate buffer  intraperitoneally. The animals were observed over a week and their blood glucose values were measured. Mice with blood glucose levels between 180 and 360 mg/dL were assigned diabetic and were used later for further studies .n = 6 each). The mice were fasted for 18 h and provided water ad libitum. Each animal serves as its own control, Group I received only glucose at dose of 2 g/kg. Groups II and III received 250 and 500 mg/kg of the ethanolic leaves extract respectively, while group IV received the isolated compound in a dose of 50 mg/kg. All the tested samples were given orally 90 min before the glucose dose . Levels of blood glucose were calculated before and subsequently at 30, 60, 120 and 240 min after the administration of glucose dose. Blood glucose was measured by glucose estimation kit.Animals were divided into four groups (n = 6 each). Group I kept as control, group II received 5 mg/kg glibenclamide as positive control, groups III and IV received the extract at doses of 250 and 500 mg/kg respectively, and group V received the isolated compound (2) at dose of 50 mg/kg. Levels of the blood glucose were measured pre and post (120 and 240 min) the treatment.The diabetic animals were divided into five groups , as following: Group I, kept as control group received daily 1% w/v sodium CMC suspension. Animals in groups II-V were hyperlipidemic and received daily cholesterol (25 mg/kg/day) in oil at 10:00 am. Group II represented the hyperlipidemic group, group III served as positive control and received lovastatin (10 mg/kg/day) at 3:00 pm. Similarly groups IV and V were given the ethanolic leaves extract at doses of 250 and 500 respectively at 3:00 pm. For a period of 30 days, the original and the final body weights and food intake of rats were monitored.The animals were grouped into five treatment categories  was assessed according to the method previously described . The finThe following formula was used to calculate the percentage inhibitory activity (I):Where A: activity without inhibitor; a: negative control in absence of inhibitor; B: activity in presence of inhibitor; and b: negative control in presence of inhibitor.Orlistat and DMSO were used as positive and negative control respectively and their activities were also tested.For the in-vitro evaluation of HMG-CoA inhibitory activity, similar concentrations range of the plant extract and isolated compound were used. Pravastatin was used as standard drug with concentrations ranged from 0.1–2.5 μg/mL according to the method previously described . HMG-CoAInhibitory activity (I %) = (∆ Absorbance control- ∆ Absorbance test/∆ Absorbance control) × 100.P values < 0.05 were considered significant. IC50 values for the DPPH radical scavenging, pancreatic lipase Inhibition and HMG-CoA inhibition assays were determined from the dose–response curves using a linear regression analysis. For in-vitro evaluation of pancreatic lipase and HMG-CoA inhibition activities, inhibition of less than 40% was considered irrelevant and was selected as a cutoff point.The results were expressed as mean + S.E.M (Standard Error Mean). Data was analyzed by GraphPad Software version 6.00 . One-Way ANOVA followed by Bonferroni’s multiple comparison tests against the control was performed. For repeated measures in glucose tolerance test, two-way ANOVA assessed the interactive and independent effects of treatment and time. Different solvents were used for leaves extraction for the flavonoid and the phenolic contents to select the safest and the most effective extracting solvent as shown in Table of C. azarolus var. eu- azarolus Maire revealed that 11 components were identified at λ=280 nm : δH 0.79, 0.98, 0.97, 1.0 and 1.2 , 0.93, , 0.94 , 1.62 , 1.38 , 2.10 , 3.32 , 5.30 .Compound 1 (ursolic acid): Molecular formula, C30 H48 O3; white powder; 20 mg; soluble in chloroform; Rf: 0.70 (System A); gave positive test for sterols and/or triterpenes; m.p., 286 °C; EI-MS m/z (% rel. Intensity) 456; 32 H50O4; white powder; 300 mg; soluble in chloroform; Rf: 0.60 (System A); gave positive test for sterols and/or triterpenes; m. p., 268–270 °C; EI-MS m/z (% rel. Intensity) 498; 1HNMR : δH 0.76, 0.85, 0.87, 1.00, 1.1 , 0.87 , 0.91 , 1.21 , 1.26 , 2.15 , 2.41 , 4.43 , 5.1. 13C NMR data for compounds 1 and 2 were recorded in Table Compound 2 (3β-O acetyl ursolic acid): Molecular formula, C14H6O8; yellow crystalline; 15 mg; soluble in methanol; Rf: 0.47 (System B); gave positive test for FeCl3; m.p. 370 °C, EI-MS m/z (% rel. Intensity) 302; colour in visible light, yellowish brown; Under UV light and under UV/ammonia, brown, UV λmax nm: CH3OH, 253, 354; 1H NMR : δH 7.49  and 7.67 .Compound 3 (ellagic acid): Molecular formula, C16H12O7, yellow powder, 35 mg; soluble in methanol; Rf: 0.50 (System C); yellowish brown in visible light; brown under UV365nm light and yellow under UV365nm/NH3, UV λmax nm: CH3OH, 256, 294sh, 358; CH3ONa, 270, 331, 407; AlCl3, 274, 300sh, 336, 440; AlCl3/HCl, 264, 300sh, 360, 402; NaOAc, 273, 323, 380; NaOAc/H3BO3, 262, 297sh, 380; 1HNMR (400 MHz. DMSO): δH 3.84 , 6.21 , 6.49 , 6.84 , 7.40 , 7.70 .Compound 4 (quercetin 3-O -β methyl ether): Molecular formula, C27H30O16; yellow powder; 20 mg; soluble in methanol; Rf: 0.38 (system B); m.p. 245 °C; brown in visible light, dull brown under UV365nm, and yellow under UV365nm/NH3; UV λmax nm: CH3OH, 260, 266 sh, 300 sh, 356; CH3ONa, 278, 327, 411; AlCl3, 270, 301sh, 430; AlCl3/HCl, 267, 298, 360 sh, 400; NaOCOCH3, 269, 325, 390; NaOAc/H3BO3, 261, 300, 385; 1HNMR, : δH 6.11 , 6.31 , 6.78 , 7.57 ; 3.33–3.67 , 4.41, 1.07 , 5.2 .Compound 5 (rutin): Molecular formula Cf: 0.22 (system B); brown in visible light, dull brown under UV365nm, and yellowish green under UV365nm/NH3; UV λmax nm: CH3OH, 267, 335; CH3ONa, 247 sh, 267, 300 sh, 385; AlCl3, 272, 300, 350, 381; AlCl3/HCl, 270, 300, 347, 380; NaOCOCH3, 254 sh, 265; 355, 385; NaOAc/H3BO3, 272, 373; 1H–NMR, : δH 7.91 , 7.03 , 6.86 , 6.51 , 6.32 , 3.14–3.53 , 4.40 , 1.07 , 5.1 . 13C NMR data for compounds 4–6 were recorded in Table Compound 6 (apigenin 7-O- β-D-glucopyranosyl (6 → 1)- α-L-rhamnopyranosyl-): yellow powder; 15 mg; soluble in methanol; R1H- and 13C–NMR spectra showed that compounds 1 and 2 were pentacyclic triterpene. Compound 1 was identified as ursolic acid while compound 2 was identified as 3β-O acetyl ursolic acid from their physical properties and their spectral data (1H–NMR and 13C–NMR) [Compounds 1 and 2 gave positive Salkoweski reactions confirming their triterpenoidal nature . 1H- and13C–NMR) , 34.1H–NMR) [The structure of compound 3 was identified as ellagic acid from its physical properties and different spectroscopic spectra (UV and1H–NMR) .1H and 13C–NMR spectra of compound 4 demonstrated a methoxy group at δH 3.84 and at δC 58.2 respectively attached at position 3; compound 4 was identified as quercetin 3-O -β methyl ether [yl ether .1H–NMR and 13C–NMR) [The structure of compound 5 was identified as rutin from its physical properties and different spectral data  .max (335 nm) of compound 6 suggested that it possesses a flavone substituted skeleton. This was confirmed from the 1H–NMR spectrum. The occurrence of two doublets signals at δH 6.51 and 6.86 (J = 2.2 Hz) indicated the presence of two meta protons at C-6 and C-8 of ring A respectively. In addition, 2 doublets appeared at δH 7.03  and at δH 7.91  indicated, H-3′, H-5’and H-2′, H-6′, respectively and one singlet at δH 6.32 for H-3. The presence of two anomeric signals at δH 4.40  and at 5.1  with the methyl group of rhamnose at δH 1.07  indicated the presence of 2 sugar units linked to C-7 position with 6 → 1 linkage. The sugar moieties were identified as glucose and rhamnose . Compound 6 could be identified as apigenin 7-O- β-D-glucopyranosyl (6 → 1)- α-L-rhamnopyranosyl (apigenin 7-O-rutinoside) [The UV λinoside) . Figure 50’s of the leaves extracts were 129.2, 140.1, 164.1 and 262.3 μg/mL for the ethanolic, methanolic, ethyl acetate and acetone respectively. While for ascorbic acid, the IC50 was calculated to be 34.6 μg/mL.DPPH free radical scavenging effects of the extracts were tested and the results are presented in Fig. n-hexane fraction and the isolated compounds, ursolic acid, 3β-O acetyl ursolic acid and quercetin 3-O- methyl ether, showed variable antimicrobial activity against all the tested pathogenic strains bacteria and fungus. On the other hand, all the tested samples exhibited variable antibacterial activities with inhibition zones ranging from 18 to 28 mm in diameter against P. aeruginosa.Results displayed in Table The isolated compound 2, 3β-O acetyl ursolic acid, demonstrated the highest growth inhibitory activity against all the tested microorganisms, followed by quercetin 3-O- methyl ether and lastly, ursolic acid.50 of the ethanolic extract of C. azarolus var. eu- azarolus Maire was safe up to 5000 mg/kg. During the observation period, no signs of morbidity or behavioral alteration in any animals’ groups were noticed.The LDp < 0.01). This marked improvement in glucose tolerance was continued over the tested time.Blood glucose levels of normal mice were significantly reduced after receiving the plant extract at different doses in a time- and dose-dependent manner as shown in Fig. p > 0.05).The basal glycaemia was 241.7 ± 1.7 mg/dl for the diabetic control mice. There was no statistical difference between the glycaemic levels of the studied groups at time 0, . A highly significant decrease of the blood glucose level was observed with 3β-O acetyl ursolic acid at the same timing intervals, p < 0.001.The anti-hyperglycemic activity of the ethanolic extract and the isolated compound (2) on the fasting blood sugar levels of diabetic mice is shown in Fig. p > 0.05). On the other hand, groups received the ethanolic leaves extract showed a significant reduction in the percentage increment in body weight in day 15 and 30 (p < 0.01) as shown in Table Compared to cholesterol induced hyperlipidemic control group (group II), lovastatin treated group showed that the reference has no effect on the body weight . Moreover, 3-β-O acetyl ursolic acid at concentrations between 80 and 200 μg/mL significantly reduced the pancreatic lipase activity with 42.4–99.6% and the IC50 was 93.6 μg/mL. On the other hand, the plant extract showed moderate inhibition of HMGCoA reductase activity at concentrations between 350 and 500 μg/mL of 42.6–60% with IC50 394.1 μg/mL compared to pravastatin that has IC50 of 0.71 μg/mL. 3-β-O acetyl ursolic acid didn’t show any significant inhibition of HMGCoA at the tested concentration.Diabetes Mellitus and other hyperglycemic disorders are complicated conditions associated with high prevalence of infection, dyslipidemia, hypertension and renal failure. The aim of this study was to find a standard plant extract that has the potential to control hyperglycemia with its associated complications, and to isolate and identify the active components that are responsible for those activities.n-butanol fractions for the first time from C. azarolus var. eu- azarolus Maire. Ursolic acid is a triterpenoidal compound that finds in medicinal herbs, other plants and foods [S. aureus, E. coli, P. aeruginosa and C. albicans [n- butanol fraction showed inhibition activity against both P. aeruginosa and E. coli. These activities are essentially linked to the phenolic constituents namely, quercetin 3-O-methyl ether, rutin and apigenin 7-O- β-D-glucopyranosyl (6 → 1)-α-L-rhamnopyranosyl. For the biological studies, doses were selected depending on the LD50 value (<1/10). Blood glucose levels of normal and diabetic mice received either ethanolic extract or 3β-O acetyl ursolic acid, were significantly decreased in a time- and dose-dependent manner. It has been previously reported that ursolic acid improves hepatic insulin resistance by stimulating the expression of peroxisome proliferator-activated receptors α (PPARα) [Results of total phenolic and flavonoid contents revealed that ethanol was the best solvent to extract both flavonoid and phenolic acids. Therefore, ethanol had been selected for further investigations. RP- HPLC analysis of the phenolics demonstrated high contents of rutin, salicylic and ellagic acids in the plant. Six compounds belonging to triterpenes and phenolic were isolated from chloroform and nd foods . Ursolicnd foods –43. Mostnd foods , 45. Thend foods –49. Hypend foods . Blood gnd foods . Diabetialbicans , 53. The (PPARα) . Moreove (PPARα) . Those e (PPARα) . Polyphe (PPARα) , 58. Suc (PPARα) . Thereby (PPARα) . InteresC. azarolus var. eu-azarolus Maire in controlling hyperglycemia with its associated complications such as infection and dyslipidemia. This multiple pharmacological profile might be due to the synergistic effect of its bioactive constituents including triterpenes, particularly ursolic acid and its acetyl derivative, and the phenolic compounds particularly, quercetin 3-O -β methyl ether, rutin and apigenin 7-O-rutinoside. This study is unique in the sense that it is the first to evaluate the antihyperglycemic and antihyperlipidemic potentialities of 3β-O acetyl ursolic acid.The current study demonstrates the efficiency of the leaves extract of


Spark plasma sintering of the SnTe nanoparticles produces nanostructured compacts that have a comparable thermoelectric performance to bulk counterparts synthesised by more time- and energy-intensive methods. This approach, combining an energy-efficient, surfactant-free solution synthesis with spark plasma sintering, provides a simple, rapid, and inexpensive route to p-type SnTe nanostructured materials.A facile one-pot aqueous solution method has been developed for the fast and straightforward synthesis of SnTe nanoparticles in more than ten gram quantities per batch. The synthesis involves boiling an alkaline Na ZT = 2SσT/κ, with S, σ, T, and κ representing the Seebeck coefficient, electrical conductivity, absolute temperature, and thermal conductivity, respectively ZT = S2σTectively , Mn 22]ZT = S2σTectively , have bealloying ,25, nanoalloying ,28,29,30alloying , and all23–873 K ,19,20,2923–873 K ,22,26. N23–873 K ,32,33. AMonoliths which are consolidated directly from metal chalcogenide nanomaterials have been demonstrated to be efficient thermoelectrics, mainly due to the enhanced phonon scattering by nanostructures ,35,36,37Herein, we demonstrate a one-pot surfactant-free aqueous solution approach for the fast and straightforward preparation of >10 g of SnTe nanoparticles, with short reaction durations. The phase-pure, surfactant-free nanoparticles can be sintered into dense compacts with a stable p-type conducting behaviour and relatively low thermal conductivity.2·2H2O  were added to 50 mL deionised water (DIW) within a two-neck round-bottom flask, to yield a transparent Na2SnO2 solution. In parallel, 10 mmol Te  and 20 mmol NaBH4  were added to 50 mL DIW within a single-neck round-bottom flask, to prepare a purple 0.2 mol·L−1 NaHTe solution:In a typical synthesis of SnTe nanoparticles, 100 mmol NaOH  and 10 mmol SnCl2SnO2 solution was heated to its boiling temperature using an oil bath, the freshly prepared 50 mL NaHTe solution was promptly injected into the solution  and the duration of the boiling process , were independently changed to investigate their effects on the synthesis. Scaled-up syntheses were performed with five-fold precursor amounts, i.e., using 500 mmol NaOH, 50 mmol SnCl2·2H2O, 50 mmol Te, and 100 mmol NaBH4 , to avoid possible reactions with ambient air.After the Naol NaBH4 c; the prol NaBH4 d, demons1 radiation, λ = 1.5406 Å). Diffraction data were collected at room temperature with a step size of 0.017 ° over 10 ° ≤ 2θ ≤ 90 ° for 1 h. The crystal structures of the synthesised products were refined via the Rietveld method against PXD data, using the GSAS and EXPGUI software packages 5 S·m−1 arations) , Mn 22]5 S·m−1 aIn summary, we have developed a simple, quick, and inexpensive one-pot solution route for synthesising SnTe nanoparticles in gram quantities (>10 g per run for a 2 h growth). Dense, nanostructured SnTe pellets have been fabricated by spark plasma sintering with a thermoelectric performance comparable to SnTe bulk materials synthesised by energy-intensive methods. As a similar solution method has already been applied to synthesise high-performance SnSe nanomaterials  and can


Aim: Docosahexaenoic acid (DHA) is known to activate the vascular large-conductance calcium-activated potassium (BK) channels and has protective effects on the cardiovascular system. However, the underlying mechanisms through which DHA activates BK channels remain unclear. In this study, we determined such mechanisms by examining the effects of different concentrations of DHA on BK channels in freshly isolated rat coronary arterial smooth muscle cells (CASMCs) using patch clamp techniques.Methods and Results: We found that BK channels are the major potassium currents activated by DHA in rat CASMCs and the effects of DHA on BK channels are concentration dependent with a bimodal distribution. At concentrations of <1 μM, DHA activated whole-cell BK currents with an EC50 of 0.24 ± 0.05 μM and the activation effects were abolished by pre-incubation with SKF525A (10 μM), a cytochrome P450 (CYP) epoxygenase inhibitor, suggesting the role of DHA-epoxide. High concentrations of DHA (1–10 μM) activated whole-cell BK currents with an EC50 of 2.38 ± 0.22 μM and the activation effects were unaltered by pre-incubation with SKF525A. Single channel studies showed that the open probabilities of BK channels were unchanged in the presence of low concentrations of DHA, while significantly increased with high concentrations of DHA. In addition, DHA induced a dose-dependent increase in cytosolic calcium concentrations with an EC50 of 0.037 ± 0.01 μM via phospholipase C (PLC)–inositol triphosphate (IP3)–Ca2+ signal pathway, and inhibition of this pathway reduced DHA-induced BK activation.Conclusion: These results suggest that DHA can activate BK channels by multiple mechanisms. Low concentration DHA-induced BK channel activation is mediated through CYP epoxygenase metabolites, while high concentration DHA can directly activate BK channels. In addition, DHA at low and high concentrations can both activate BK channels by elevated cytosolic calcium through the PLC–IP3–Ca2+ signal pathway. Both EPfunction . DHA hasfunction . In addifunction .+) channels in coronary arterial smooth muscle cells (CASMCs): the voltage-gated potassium (Kv) channels, the ATP-sensitive K+ (KATP) channels, the large-conductance (BK), the intermediate-conductance (IK), and the small-conductance (SK) calcium (Ca2+)-activated K+ channels –Ca2+ signal pathway. These findings indicate that DHA activates BK channels through multiple mechanisms.In the present study, we found that the profile of BK channel activation by DHA has a bimodal distribution. We therefore hypothesized that the mechanisms of BK channel activation by DHA are different at low and high concentrations. To test this hypothesis, we determined the mechanisms of BK channel activation by DHA at different concentrations using patch clamp techniques and cytosolic calcium fluorescent ratio measurements. We found that activation of BK channels by DHA at concentrations >1 μM is independent of CYP epoxygenase activity. In addition, DHA at low and high concentrations can both activate BK channels by elevated cytosolic calcium via the phospholipase C (PLC)–inositol triphosphate  were obtained from the Harlan Laboratories  and from Shanghai SLAC Laboratory Animal Co., Ltd. . This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011). The protocol was approved by the Institutional Animal Care and Use Committee of Mayo Foundation and the Committee on Animal Care of Wuxi People’s Hospital affiliated to Nanjing Medical University.2 0.05, MgCl2 1.0, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10.0, and glucose 10.0 at pH 7.4 with NaOH. Rat coronary arteries  were carefully dissected free of surrounding myocardium and connective tissue. Freshly isolated rat coronary arteries were incubated with 1.0 ml physiological saline solution containing bovine serum albumin  for 10 min at 37°C in a shaking water bath followed by digestion in 1.0 ml saline solution containing 1.5 mg papain, 1.0 mg dithiothreitol, and 0.1% bovine serum albumin at 37°C for 10 min. The vessels were further treated with 1.0 ml saline solution containing 1.0 mg collagenase, 1.0 mg trypsin inhibitor, 0.25 mg elastase, and 0.1% bovine serum albumin at 37°C for another 10 min and then washed three times with 1.0 ml aliquots of saline solution. The vessels were gently triturated with a fire-polished glass pipette until cells were completely dissociated.Rat CASMCs were dissociated enzymatically, as described previously . Briefly+ currents were recorded from a holding potential (HP) of -60 mV with pulses of 100 ms duration to testing potentials (TPs) from -40 to +160 mV in 10 mV increments. Pipette resistance was 2.0–4.0 MΩ when filled with the pipette solution which contained (in millimolar) KCl 140.0, MgCl2 0.5, Na2ATP 5.0, Na2GTP 0.5, HEPES 10.0, ethylene glycol tetraacetic acid (EGTA) 1.0, CaCl2 0.465 (∼200 nM free Ca2+) at pH 7.35. The bath solution for whole-cell recordings contained (in millimolar) NaCl 145.0, KCl 5.6, MgCl2 1.0, CaCl2 0.5, HEPES 10.0, and glucose 10.0 at pH 7.4. To investigate the activation effects of DHA on different K+ channels, various K+ channel blockers were applied after baseline K+ current recordings were obtained. In brief, we used tetraethylammonium  for total K+ currents, iberiotoxin  for BK channels, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole  for IK channels, apamin  for SK channels, 4-aminopyridine  for Kv channels, and glyburide  for KATP channels.Whole-cell and inside-out single BK currents were recorded from freshly isolated rat CASMCs as we have previously described  using an2+ sensitivity. Channel open probabilities (NPO) were calculated using Clampfit 10.2 software . Pipette resistance was 5–10 MΩ when filled with the pipette solution which contained (in millimolar) KCl 140, CaCl2 1, MgCl2 1, HEPES 10, and EGTA 1 at pH 7.4. The bath solution contained (in millimolar) KCl 140, MgCl2 1, EGTA 1, HEPES 10, CaCl2 0.816 (1 μM free Ca2+) at pH 7.35.Inside-out single BK channel currents were elicited at +60 mV. The BK channels were identified by their unitary conductance, voltage sensitivity, and Ca2HPO4•12H2O 0.363, KH2PO4 0.441, and NaHCO3 4.17 (in millimolar) for 30 min. The cells were then washed and superfused with Hanks’ solution in a perfusion bath mounted on the stage of an inverted microscope. Fluorescence intensities were recorded with excitation wavelengths 340 and 380 nm, and emission wavelength 510 nm. Fluorescent intensity ratios of F340/F380 were calculated, and the changes of fluorescent intensity ratios were used to detect the changes of cytosolic calcium concentrations in this study as previously reported  as we have previously reported . Brieflyreported .All chemicals were obtained from Sigma-Aldrich Co. , except DHA was purchased from Cayman Chemical  and SKF525A from BIOMOL . In this study, we defined low concentrations of DHA as <1 μM and high concentrations as >1 μM.t-test was used to compare data between two groups. One-way ANOVA followed by contrast testing was used to compare data from multiple groups. Statistical significance was defined as P < 0.05.Data were presented as mean ± SEM. Student’s + currents were significantly increased by 5 μM DHA. Upon exposure to 5 μM DHA, the K+ currents were enhanced several folds from baseline, and these effects were reversed by DHA washout . The time course of the effects of 5 μM DHA on K+ currents is shown in Figure 1B. The current–voltage (I–V) relationships of K+ currents at baseline, with DHA, and after washout are shown in Figure 1C.In our previous study, we reported that BK channel activation in rat CASMCs by DHA at 1 μM was dependent on CYP epoxygenase activity . In this+ channels in rat CASMCs, and the BK and Kv currents are the major constituents  with no inhibitors, 13.9 ± 2.7 and 14.1 ± 3.2 pA/pF  in the presence of TEA (10 mM), 25.1 ± 5.6 and 89.1 ± 29.2 pA/pF  in the presence of IBTX (100 nM), 55.8 ± 9.2 and 380.6 ± 125.5 pA/pF  in the presence of TRAM-34 (200 nM), 56.5 ± 8.7 and 345.6 ± 110.1 pA/pF  in the presence of 4AP (5 mM), 35.3 ± 9.9 and 326.6 ± 97.6 pA/pF  in the presence of APA (1 μM), 68.6 ± 6.8 and 349.6 ± 115.9 pA/pF  in the presence of GLY (10 μM), 23.0 ± 7.2 and 81.6 ± 23.7 pA/pF  in the presence of IBTX (100 nM) plus TRAM-34 (200 nM) and APA (1 μM), and 20.9 ± 3.8 and 73.9 ± 21.9 pA/pF  in the presence of IBTX (100 nM) plus 4AP (5 mM). The increasement of K+ currents was much smaller in the presence of IBTX than other blockers (P < 0.05), suggesting that BK currents are the major constituents of K+ currents activated by DHA in rat CASMCs. Group data are summarized in Figure 1E. However, after blocking KCa (in the presence of IBTX plus TRAM-34 and APA) or BK and Kv channels (in the presence of IBTX plus 4AP), there was still a small response to DHA, which may also activate chloride channels or other non-specific cation channels, such as transient receptor potential channels, etc.Since there are different types of Ktituents , we needFigure 2A . As we have previously reported . The DHA–BK current concentration–response curve is shown in Figure 2B. It is best described by curve fitting showing a double sigmoidal pattern with two plateaus . Two half-maximal effective concentrations (EC50) are indicated: with a high potency of 0.24 ± 0.05 μM at low concentrations of DHA (0–1.0 μM) and a low potency EC50 of 2.38 ± 0.22 μM at high DHA concentrations (1.0–10 μM) . This behavior of DHA on BK channel activation is unusual. These results suggested that DHA activates BK channels via different mechanisms at low and high concentrations.Since BK channels are the major targets of DHA activation, further experiments were performed with 5 mM 4AP, 1 μM APA, and 200 nM TRAM-34 in the bath solution to block the Kv, SK, and IK channels. Under such conditions, BK currents were activated by DHA in a concentration-dependent manner. Representative current traces were displayed in reported , DHA hadn = 6 cells, P < 0.05, Figure 2C). These findings suggested that activation of BK channels by DHA at low concentrations is dependent on CYP epoxygenase activity but not at higher concentrations. In the presence of SKF525A, DHA activated BK channels with an EC50 of 3.31 ± 0.59 μM .We have previously reported that activation of BK channels by DHA at <1 μM is dependent on CYP epoxygenase activity and the activation effects of DHA on BK channels can be blocked by the CYP epoxygenase inhibitors SKF525A or MS-PPOH . To deten = 5 cells, P > 0.05 vs. no DHA). In the presence of 3, 5, and 10 μM DHA, BK channel open probabilities were 0.232 ± 0.027, 0.582 ± 0.041, and 0.606 ± 0.066, respectively . These results suggested that DHA had no direct effects on BK channels at concentrations of <1 μM but is a robust direct BK channel activator at >1 μM with an EC50 of 3.37 ± 0.10 μM .Single BK channel currents were recorded from freshly isolated rat CASMCs in inside-out configuration. With 0, 0.01, 0.1, 0.3, and 1 μM DHA in the bath solution and in the presence of 1 μM free calcium, BK channel open probabilities at a membrane potential of +60 mV were 0.075 ± 0.019, 0.071 ± 0.020, 0.067 ± 0.019, 0.087 ± 0.026, and 0.098 ± 0.023, respectively  at 0.001–0.01 μM, but fluorescence intensity ratios were increased on exposure to DHA at higher concentrations with an EC50 of 0.037 ± 0.01 μM  .It is well-known that BK channels in vascular smooth muscle cells are activated by an elevation of cytosolic calcium concentration. To determine whether DHA activates BK channels by elevated cytosolic calcium, we recorded signals from Ca3 pathway on the increase of [Ca2+]i by DHA, rat CASMCs were treated with U73122, an inhibitor of PLC and with 2-APB, a IP3 receptor blocker. After incubating with U73122, the fluorescence intensity ratios were significantly blunted upon exposure to DHA compared with controls  . Similarly, after incubating with 2-APB, the fluorescence intensity ratios were significantly blunted on exposure to DHA compared with controls  . These results indicated that DHA increases intracellular Ca2+ in rat CASMCs through PLC–IP3-dependent signal pathways. Hence, increase in intracellular Ca2+ is another potential mechanism through which DHA mediates the activation of coronary BK channels.To determine the role of the PLC–IP3 signal pathway on BK activation by DHA, BK currents were recorded at baseline and after exposure to 1 and 5 μM of DHA in rat CASMCs  with and without U73122 and 2-APB. Baseline BK current densities were not affected by incubation with U73122 and 2-APB  but the ability of DHA at 1 and 5 μM in BK channel activation in CASMCs was significantly diminished  . These results suggest that the PLC–IP3 signaling pathway is important in mediating BK channel activation by DHA in vascular smooth muscle.To further confirm the role of PLC–IP+ channels in CASMCs, but predominantly BK channels. Second, activation of BK channels by DHA has a double sigmoidal pattern with two plateaus. Low concentrations of DHA-induced BK channels activation are dependent on the activity of the CYP pathway, while high concentrations of DHA directly activate BK channels. Third, DHA increases the cytosolic calcium of rat CASMCs in a dose-dependent manner. Fourth, DHA can activate BK channels via the PLC–IP3–Ca2+ signal pathway. These important novel findings indicate that DHA activates the coronary BK channels through multiple mechanisms.In this study, we have made several important findings. First, DHA activates several K+ channels and the role for BK channels is insignificant. In this study, we found that DHA potently activated total K+ currents but this effect was very weak in the presence of IBTX, a specific BK channel blocker, indicating that the activation of K+ currents in CASMCs by DHA is mainly mediated through BK channels.Large-conductance calcium-activated potassium channels are abundantly expressed in vascular smooth muscle cells , and thein vivo (We have previously reported that the effects of DHA at <1 μM on BK channel activation were dependent on CYP epoxygenase activity and abolished by pre-incubation with a CYP inhibitor (SKF525A or MS-PPOH) in rat CASMCs . Recentlin vivo . Ye et ain vivo .50 as shown in Figure 2. The effects of DHA epoxides are potent activators of BK channels but their effects appear to be saturated at sub-micromolar concentrations. Hence, the dietary consumption of DHA represents an important source of BK channel activators help preserve cardiovascular function including blood pressure control and vital tissue perfusion.Since human plasma concentrations of DHA is in the micromolar range , we foun2+ sensors of BK channels  channels –PLC–IP3 pathway consequently activating BK channels  in CASMCs . The STO2+ . In humachannels . Thus, D37 cells . In thischannels . The oxyr U73122 . DHA diln by DHA . To furtFigure 7). DHA-induced BK channel activation at low concentrations (<1 μM) is mediated through CYP epoxygenase metabolites, while DHA at high concentrations (>1 μM) directly activates BK channels, probably by binding to the S6 segment in the α-subunit. In addition, DHA can activate BK channels through elevated cytosolic calcium via the PLC–IP3–Ca2+ mechanism. BK channel should be considered as a novel therapeutic target for the prevention and/or treatment of vascular abnormalities in diabetes, hypertension, and other vasculopathic conditions.To summarize, our results demonstrated that DHA can activate BK channels through multiple mechanisms (R-XW, L-LQ, and M-QS were involved in the experiment design. L-LQ, M-QS, R-XW, TL, YW, YJ, XT, X-XZ, and WW performed the experiments. L-LQ, S-PD, X-YL, and R-XW analyzed the data. L-LQ and M-QS wrote the manuscript. R-XW, TL, H-CL, QC, MP, FY, and D-MZ edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thermoactinospora rubra YIM 77501T is an aerobic, Gram-positive, spore-forming and cellulose degrading thermophilic actinomycete isolated from a sandy soil sample of a volcano. Its growth temperature range is 28–60°C. The genomic sequence of this strain revealed that there are 27 cellulase genes belonging to six glycoside hydrolase families. To understand the strategy that this strain uses to utilize carbon sources such as cellulose at different temperatures, comparative transcriptomics analysis of T. rubra YIM 77501T was performed by growing it with cellulose (CMC) and without cellulose (replaced with glucose) at 30, 40, and 50°C, respectively. Transcriptomic analyses showed four cellulase genes  were up-regulated at 30, 40, and 50°C. The rate of gene expression of TrBG2, TrBG3, TrBG4, and ThrCel6B were 50°C > 30°C > 40°C. One cellulase gene (TrBG1) and two cellulase genes (TrBG5 and ThrCel6A) were up-regulated only at 30 and 50°C, respectively. These up-regulated cellulase genes were cloned and expressed in Escherichia coli. The enzymatic properties of up-regulated cellulases showed a variety of responses to temperature. Special up-regulated cellulases TrBG1 and ThrCel6A displayed temperature acclimation for each growth condition. These expression patterns revealed that a hybrid strategy was used by T. rubra to utilize carbon sources at different temperatures. This study provides genomic, transcriptomics, and experimental data useful for understanding how microorganisms respond to environmental changes and their application in enhancing cellulose hydrolysis for animal feed and bioenergy production. Paenibacillus sp. IHB B 3084 shows optimum activity below 45°C  medium to stationary phase at different temperatures showed that the cellulase activity of the culture at 30 and 50°C were higher than culture at 40°C  activity showed that the sample from 30°C had higher activity at 30–45°C, and the sample from 50°C had higher activity at 50–70°C. These clues clearly indicate that T. rubra could express cellulases with different enzymatic characteristics to digest cellulose as carbon source at different temperatures.e Figure . The cruC Figure . FurtherT. rubra YIM 77501T was sequenced. The gene predication and function annotation revealed that there were 408 glycoside-hydrolase-encoding genes. The transcriptomes of T. rubra under different culture conditions  were profiled as well. The cellulase genes, which were significantly up-regulated in the presence of CMC under different temperatures, were cloned and expressed heterogenetically. The enzymatic properties of these cellulases were characterized. This study shed light on the strategy of strain T. rubra for carbon source acquisition at different temperatures.In this study, the draft genome of T. rubra YIM 77501T (= DSM 45614T = CCTCC AA 2011014T) was cultured on R2A medium  at 50°C. Genomic DNA was purified from 100 ml of mid exponential phase R2A cultures using a MasterPure Gram-Positive DNA Purification kit (Epicentre MGP04100) following the standard DNA isolation procedure recommended by the manufacturer with modifications , the clean reads with high quality were assembled using de Bruijn graphs in SOAP de novo v.1.05 (http://www.seekbio.com/soft/2754.html) with the K-mer parameter set to 41  was used to align these reads against the de novo scaffolds to map reads and account for single nucleotide errors  and DDBJ/ENA/GenBank (Accession number: MSZZ00000000). Carbohydrate-active enzymes (CAZymes) of T. rubra were determined using the CAZymes Analysis Toolkit : yeast extract, 0.5 g; peptone, 0.5 g; casein acid hydrolysates, 0.5 g; sodium pyruvate, 0.5 g; K2HPO4, 0.3 g; MgSO4, 0.024 g; with 2 g/l CMC sodium as the test sample (R2A-CMC medium) and with 2 g/l glucose as control (R2A-glucose medium), with the pH adjusted to 7.0 using KOH. The colony of T. rubra was inoculated to R2A-CMC and R2A-glucose media. The growth curves under different conditions were determined .d Figure . After iT. rubra using Bowtie 2.2.3 . Raw reads of fastq files were first processed through in-house Perl scripts. In this step, clean reads were utilized by removing reads containing adapter, reads containing poly-N and low quality reads from raw data. At the same time, the Q20, Q30, and GC contents of the clean data were calculated. All the following analyses were based on clean data with high quality. The reads from different conditions were mapped to the whole-genome of strain p-values were adjusted using the Benjamini and Hochberg approach for controlling false discovery rate. Genes with an adjusted p < 0.05 found by DESeq were assigned as differentially expressed  at the same temperature  were performed using the DESeq R package (1.18.0). DESeq provided statistical methods for determining differential expression in digital gene expression data using a model based on a negative binomial distribution. The resulting T. rubra genomic DNA. Chromosomal DNA of T. rubra was isolated using the Ezup Bacteria DNA Kit  according to the manufacturer's instructions. Base on genome sequences, primers of cellulase genes (Table http://www.bioprocessonline.com/doc/primer-premier-5-design-program-0001). The complete ORFs of cellulase genes were amplified by PCR using the TransStar FastPfu Fly DNA Polymerase . Amplification was performed for 34 cycles of 98°C for 20 s, 65°C for 20 s, and 72°C for 1 min, 72°C for 5 min with initial 2 min denaturation at 98°C. PCR products were ligated into pEASY-Blunt E1 vector  and transformed into Escherichia coli DH5α. After sequencing verification, the entire cellulase gene was confirmed; positive recombinant vectors were transformed into E. coli BL21 (DE3) for cellulase gene expression.The full-length sequences of cellulase genes were amplified from es Table  were desTransformants were cultured overnight in LB culture medium with 100 μg/ml ampicillin at 37°C and 220 rpm. Then, 1 ml of the cells was added to 100 ml LB medium at 25°C and 220 rpm. During cultivation, isopropyl β-D-1-thiogalactoside (IPTG) was added to a final concentration of 1 mM at mid-exponential phase (OD600 ≈ 0.6) and followed by further incubation 8 h at 25°C with 220 rpm. Cells were harvested by centrifugation at 4,000 × g and suspended in 20 ml PBS buffer (pH 8.0).After ultrasonic cell disintegration and centrifugation at 12,000 × g for 30 min at 4°C, cell-free extracts were applied to a Ni-chelating affinity column  because the proteins possess an N-terminal His-tag. Then, the extract was washed with five column volumes of buffer A , followed by 10 column volumes of buffer A with 20 mM imidazole, pH 8.0, and was eluted with buffer A with 200 mM imidazole, pH 8.0. The eluted protein was used for enzyme characterization.2CO3  were distributed into 58 families, 226 carbohydrate-binding modules (CBMs) were distributed into 20 families, 20 polysaccharide lyases (PLs) were distributed into 7 families, 109 carbohydrate esterases (CEs) were distributed into 10 families, and 339 glycosyl transferases (GTs) were distributed into 26 families, are encoded in the genome of T. rubra cultured on R2A-glucose and R2A-CMC media under different temperatures  were examined using transcriptome sequencing. Finally, there are 406.48 million clean reads and 50.81 GB of RNA-seq data in treated and control strains after quality filtering . More than 99% of the clean reads were mapped to the T. rubra YIM 77501T genome. The GC content of clean reads is within the scope of 68.2–69.2% for all samples  were up-regulated at 30, 40, and 50°C (p < 0.01). The rates of gene expression of TrBG2, TrBG3, TrBG4, and ThrCel6B were relatively higher at 50°C; on the contrary, they were relatively lower at 40°C. One cellulase gene (TrBG1) was especially up-regulated at 30°C (p < 0.01); two cellulase genes (TrBG5 and ThrCel6A) were merely up-regulated at 50°C  at 30, 40, and 50°C, respectively. In these genes, seven up-regulated cellulase genes belonging to GH1, GH3, and GH6 were detected, comparing the cellulase genes expressions of CMC cultures with glucose cultures. This means that not all cellulase genes expressed during our culture conditions and the presence of CMC were no need for expression of some cellulase genes. Comparing the cellulase genes expressions of CMC cultures with glucose cultures at same temperature, four cellulase genes  at 40°C in R2A-CMC and R2A-glucose media, respectively; 7,345 and 7,228 genes were expressed (FPKM > 1) at 50°C in R2A-CMC and R2A-glucose media, respectively, , when T. rubra was cultured at 30 and 50°C as compared at 40°C. The genes 2,725 and 2,750, 2,231 and 2,543, 2,835 and 2,852 showed high expression (FPKM > 60) when T. rubra was cultured at 30, 40, and 50°C on R2A-CMC and R2A-glucose media, respectively  when , Figure . The exply Table .T. rubra was cultured at different temperatures in the same media (R2A-CMC or R2A-glucose). Comparing 30°C with 40°C, 4,473 genes  were significant differentially expressed in R2A-CMC media and 3,894 genes  were significant differentially expressed in R2A-glucose media. Comparing 30°C with 50°C, 4,918 genes  were significant differentially expressed in R2A-CMC medium and 4,988 genes  were significant differentially expressed in R2A-glucose medium. Comparing 40°C with 50°C, 4,293 genes  were significant differentially expressed in R2A-CMC medium and 3,760 genes  were significant differentially expressed in R2A-glucose medium  were found at 30°C, 3,560 genes  were found at 40°C, and 2,869 genes  were found at 50°C. Among these differentially expressed genes at different temperatures, 1,278 genes were up- and down- regulated at all temperatures, and 732, 504, and 577 genes were specifically expressed at 30, 40, and 50°C, respectively . TrBG5 and ThrCel6A showed high relative enzyme activity at 50°C  after incubating at 50°C for 6 h  after incubating at 50°C for 3 h. These results revealed TrBG1 and TrBG5 may be unstable at 50°C T. rubra, suggesting that this strain can convert cellulose as its own carbon source into biomass. Our results demonstrated that strain T. rubra has many choices of enzymes available to degrade cellulose at different temperatures.Research has shown that some potential functions of microorganisms, such as degradation ability of carbon sources, implemented by special functional genes can be predicted based on genomic data analysis . The intergenic region may play a regulatory function in gene expression for some microorganisms  when T. rubra was cultured both at 30 and 50°Ccompared with at 40°C. This could be attributed to 30 and 50°C being nearer the growth temperature range (28–60°C) of T. rubra  exist in all organisms and are important for stress tolerance  for microorganisms  was up-regulated compared with culturing at 30 and 40°C . These results indicated that T. rubra may need to express more genes to live at a lower temperature. Global transcriptome analysis of Lactococcus garvieae strains also found more up-regulated genes at 18°C than at 37°C   to utilize carbon sources at different temperatures. Comparing CMC cultures with glucose cultures, the expression of cellulase genes showed different patterns and levels at different temperatures. Four cellulase genes  appeared to be up-regulated at three temperature conditions significantly, and one (TrBG1) and two (TrBG5 and ThrCel6A) cellulase genes were significantly up-regulated only at 30 and 50°C, respectively. Moreover, the expression levels of up-regulated cellulase genes  at 30 and 50°Cwere higher than at 40°C. These results correspond with cellulase activity of culture supernatants  at 30°C and were not stable at 50°C after 6 h. Therefore, in light of evolution, extra cellulases may appear at lower temperatures (30°C) or higher temperatures (50°C). TrBG1is a special up-regulated cellulase gene at 30°C, and it has high (>50%) relative enzyme activity at 30°C. ThrCel6A is also a special up-regulated cellulase gene at 50°C and was stable at 50°C after 6 h. TrBG5 is a special up-regulated cellulase gene at 50°C, and its optimal temperature was 50°C. However, no signal peptide was predicted by using SignalP 4.1 Server  and without cellulose (replace with glucose) at 30, 40, and 50°Cwere studied. The differences of the transcriptome analysis approach revealed that a hybrid strategy was used by T. rubra to utilize carbon sources at different temperatures. Higher levels and more numbers of cellulase genes were up-regulated at lower (30°C) or higher (50°C) temperatures than at a middle temperature (40°C). The enzymatic properties of up-regulated cellulase genes showed a variety of responses to temperature. Special up-regulated cellulases TrBG1 and ThrCel6A displayed temperature acclimation. These results may mean T. rubra has evolved an economical and efficient strategy to utilize carbon sources at different temperatures. This study provides genomic, transcriptomics, and experimental data useful for understanding how microorganisms respond to environmental changes and for their application in enhancing cellulose hydrolysis for animal feed and bioenergy production.To understand the strategies of carbon source acquisition, such as cellulose, in the environment at different temperatures for microorganisms, comparative transcriptomics of WL, YY, and XZ designed research and project outline. YY, ZJ, and WX performed growth and genome sequencing. XZ, EZ, and FZ performed transcriptome sample preparation and sequencing. QH, LL, and WH provided the gene cloning, expression and purification of cellulases. ZM, FZ, and YY performed enzyme assays and protein assays. WL, YY, and XZ drafted the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cortical circuits are profoundly shaped by experience during postnatal development. The consequences of altered vision during the critical period for ocular dominance plasticity have been extensively studied in rodent primary visual cortex (V1). However, little is known about how eye opening, a naturally occurring event, influences the maturation of cortical microcircuits. Here we used a combination of slice electrophysiology and immunohistochemistry in rat V1 to ask whether manipulating the time of eye opening for 3 or 7 d affects cortical excitatory and inhibitory synaptic transmission onto excitatory neurons uniformly across layers or induces laminar-specific effects. We report that binocular delayed eye opening for 3 d showed similar reductions of excitatory and inhibitory synaptic transmission in layers 2/3, 4, and 5. Synaptic transmission recovered to age-matched control levels if the delay was prolonged to 7 d, suggesting that these changes were dependent on binocular delay duration. Conversely, laminar-specific and long-lasting effects were observed if eye opening was delayed unilaterally. Our data indicate that pyramidal neurons located in different cortical laminae have distinct sensitivity to altered sensory drive; our data also strongly suggest that experience plays a fundamental role in not only the maturation of synaptic transmission, but also its coordination across cortical layers. Studies in patients born with bilateral or unilateral cataracts, delaying the onset of vision, have reported profoundly altered visual function. This effect is thought to depend on altered maturation of visual cortical circuits, although the mechanisms for these changes are currently unknown. We report significant differences in the effect of binocular and monocular delayed eye opening on excitatory and inhibitory synaptic transmission onto excitatory neurons in rat V1. Binocular delays had transient, but similar, effects across layers, whereas monocular delays showed long-lasting, laminar-specific changes. Our data suggest that differences in coordination of synaptic transmission across visual cortical layers at the time of eye opening may contribute to the distinct functional effects of binocular or monocular delays in early vision in patients.Cortical layers in primary visual cortex (V1) form recurrent microcircuits activated by different sets of inputs . It is wHere we used brief (3-d) and long (7-d) delays in binocular and monocular eye opening to shift the onset of vision and assess the effect of these manipulations on the E/I balance of pyramidal neurons located in all layers of V1. Our analysis straddled the developmental window between eye opening [postnatal day 14 (P14)] and the peak of the critical period (P30). Immunohistochemistry and electrophysiology were used to assess the laminar distribution of two distinct populations of inhibitory neurons and determine the E/I ratio of synaptic currents onto pyramidal neurons.+ and SST+ inhibitory neurons was not sensitive to manipulations of visual drive, synaptic transmission was significantly affected. A 3-d delay in binocular eye opening (bDEO) decreased excitatory drive in L2/3, L4, and L5 and prevented the age-dependent increase in inhibition in L2/3, 4, and 5. Interestingly, if eye opening was delayed binocularly for 7 d, synaptic transmission in all layers was comparable to that of age-matched littermates, suggesting the engagement of compensatory mechanisms. Monocular delayed eye opening (mDEO) had profoundly different effects from bDEO. A 3-d mDEO induced laminar-specific changes in excitatory and inhibitory drive onto pyramidal neurons, with significant shifts in E/I balance only in L2/3 and L4. A longer mDEO induced additional changes that further disrupted coordination across layers. Together, our data indicate that laminar circuits within neocortex show distinct patterns of maturation that are coordinated by binocular visual drive. Although symmetric delays in the onset of vision reduce synaptic drive onto pyramidal neurons without affecting the E/I balance, asymmetric delays alter the E/I balance and disrupt coordination across layers, unveiling layer-specific mechanisms of refinement. Our results indicate that the laminar difference in sensitivity to altered visual drive of local circuits in V1 should be taken into consideration when devising therapeutic approaches for the recovery of healthy visual function.We report extensive layer-specific differences in postnatal maturation of synaptic transmission and in the laminar distribution of one class of inhibitory neurons during normal postnatal development. Although the prevalence of PVAll experimental procedures were approved by the Institutional Animal Care and Use Committee of the authors’ institution and followed the guidelines of the National Institutes of Health.m phosphate buffer at 4°C . Brains were then dissected out and postfixed overnight in 4% PFA. During the following days, brains were cryoprotected with 30% sucrose and embedded in Tissue Tek OCT. Coronal slices (50 µm) containing V1 were prepared with a cryostat (MICROM HM505SE) and collected in PBS (0.1 m). Double immunostaining for parvalbumin (PV+) and somatostatin (SST+) and single immunostaining for the vesicular glutamate transported type 2  were performed in parallel on alternate slices. Slices were incubated overnight in Tris-buffered saline with Tween (TBST) solution containing the primary antibodies , then immunoreacted with secondary antibodies (1:200): Alexa Fluor 594 goat anti-mouse  and Alexa Fluor 488 goat anti-rabbit . To label VGluT2-expressing terminals, slices were immunostained with an anti-VGLUT2 antibody , followed by incubation in the secondary antibody Alexa Fluor 488 goat anti-rabbit . VGluT2 labeling was used to facilitate the identification of L4 within V1m  were deeply anesthetized with an intraperitoneal injection of a mixture of 70 mg/kg ketamine, 3.5 mg/kg xylazine hydrochloride, and 0.7 mg/kg acepromazine maleate, then transcardially perfused with PBS, followed by 4% paraformaldehyde (PFA) in 0.1 thin V1m A, B. AllFor each animal, we analyzed 10 slices taken every 100 µm to ensure sampling of V1m at different levels along the rostro-caudal axis. Images containing the monocular region of V1m were acquired with a confocal laser-scanning microscope (Olympus Fluoview 1000) using a 10× objective. V1m was identified using the coordinates from the atlas for the rat brain  correcte+ neurons within each layer was quantified using the plugin Image-based Tool for Counting Nuclei (ITCN). Once the diameter of a cell and the minimum distance between cells was chosen, it was maintained constant to analyze all acquired images. All images were acquired using a 10× objective and the same microscope settings, so that the measuring parameters were constant across animals. To quantify the percentage of PV+ or SST+ neurons in each layer of V1m, we used the plugin cell counter. Cell number was expressed as percentage of Nissl-PV+ (or SST+) neurons.Projection images were made in ImageJ. We first outlined regions of interest (ROIs) containing each layer in ImageJ. The number of NisslEye opening was delayed either with binocular eyelid suture (bDEO) or monocular eyelid suture (mDEO). Both procedures were started before the naturally occurring eye opening at P13–P14 and maintained for 3 or 7 d . To perfm): 126 NaCl, 3 KCl, 25 NaHCO3, 1 NaHPO4, 2 MgSO4, 2 CaCl2, 14 dextrose, pH 7.4, when bubbled with mixed 95% CO2 and 5% O2. For recordings, a single visual cortical slice was transferred to the recording chamber and perfused with standard ACSF. Recordings were performed at room temperature. Visually guided whole-cell recordings were obtained using an IR-DIC microscope (Olympus BX51). Recordings were performed using borosilicate glass pipettes with resistance of 3–5 MΩ and filled with an intracellular solution containing (mm): 100 Cs-sulfate, 20 KCl, 10 Hepes, 4 Mg-ATP, 0.3 Na-GTP, 10 phosphocreatine, and 0.4% biocytin. Osmolarity was adjusted to 295 mOsm with sucrose. Spontaneous EPSCs (sEPSCs) were recorded by holding the neurons at the reversal potential for GABAA-mediated currents (–50 mV), and spontaneous IPSCs (sIPSCs) were recorded at the reversal potential for AMPA–NMDA-mediated currents (10 mV). To assess the quality of the recordings, input resistance was monitored throughout recordings. Neurons with >20% change in input resistance were not included in the analysis. Patch-clamp recordings were obtained from pyramidal neurons in L2/3, L4, L5, and L6.Long-Evans rats of both sexes were anesthetized with isoflurane and decapitated. Acute coronal slices (300 µm) containing the monocular portion of V1 (V1m) were prepared as described . To idenpost hoc biocytin staining. Briefly, slices were fixed for at least 48 h in PBS (pH 7.4) containing 4% PFA. Thereafter, slices were transferred into PBS and incubated overnight with streptavidin Alexa Fluor 594 (1:1000) and fluorescent Nissl counterstaining 435/455 . A confocal laser-scanning microscope (Olympus Fluoview 1000) was used to acquire images of recorded neurons and assess their morphology and laminar location. After the confocal imaging procedures, slices were unmounted and developed with diaminobenzidine to reconstruct their entire morphology in bright field using the software Neurolucida (MicroBrightField).Pyramidal cell morphology and laminar location of all neurons included in this study were confirmed by t test and one-way ANOVA) and nonparametric tests [Mann-Whitney (MW) U test and Kruskal-Wallis (KW) ANOVA] after assessment of normality with the Shapiro-Wilk test. To determine differences across ages and layers, we used KW ANOVA followed by post hoc MW U test. Bonferroni correction was used to correct for multiple comparisons ; therefore only p values <0.008 were considered significant in these conditions. For comparisons between 2 experimental conditions, p values <0.05 were considered significant. Differences in the cumulative distributions were assessed using the Kolmogorov-Smirnov (KS) test.All data are presented as average ± standard error of the mean (SEM) for the number of neurons indicated. In this study, we used parametric (+) and somatostatin expressing (SST+). In addition, we measured excitatory and inhibitory synaptic drive onto pyramidal neurons grouped by laminar location. To assess how the time of eye opening contributes to the maturation of excitatory and inhibitory inputs onto V1 pyramidal neurons, we used two different manipulations to alter the onset of visual drive: bilateral delayed eye opening by binocular eyelid suture (bDEO) and unilateral delayed eye opening by monocular eyelid suture (mDEO).The aim of this work was to determine how the onset of visual experience regulates excitatory and inhibitory drive across cortical layers in the rat primary visual cortex (V1). We straddled the period from eye opening (P14) to the peak of the critical period for visual cortical plasticity P30;  and quan+ and SST+) neurons, are similarly distributed across cortical layers from the time of eye opening. To address this, we used immunohistochemistry and confocal microscopy and quantified number and location of PV+ and SST+ inhibitory neurons in the monocular region of V1 , whereas at P17, they were detectable in all layers. The density of PV+ neurons in each layer remained stable after P17 Before assessing the effects of eye opening timing on the V1 cortical circuit, we needed to establish the pattern of maturation of excitatory and inhibitory synaptic drive in naive animals. Several studies showed that inhibitory synapses undergo a process of maturation during a developmental window that occurs between eye opening and the peak of the critical period for visual cortical plasticity . Many as V1 V1m; A. We use V1 V1m; B. Immuno V1 V1m; , there w V1 V1m; C. Analyse groups F. As pree groups  at P14, fter P17 F. . Only p values <0.008 are considered significant owing to correction for multiple comparisons. In summary, these data show that from the time of eye opening to the peak of the critical period, synaptic drive onto pyramidal neurons in V1m differed significantly across layers. In L2/3 and L5, inhibition was dominant in all age groups, whereas it transitioned from dominant excitation to dominant inhibition in L4 and from balanced to inhibition-shifted in L6. The distinct starting points converge on a comparable inhibition-dominated state over the course of the 2 wks after eye opening  of pyramidal neurons changes during development and found a progressive decrease in L2/3, L4, and L6, but not L5 (in (MΩ) in L2/3: P14, 123 ± 8, n = 14; P17, 79 ± 4, n = 11; P21, 90 ± 7, n = 10; P30, 65 ± 3, n = 12; KW ANOVA: p < 0.001; MW test: P14 vs. P17, p < 0.001; P14 vs. P21, p = 0.03; P14 vs. P30, p < 0.001; P17 vs. P21, p = 0.09; P17 vs. P30, p = 0.01; P21 vs. P30, p = 0.009; in L4: P14, 126 ± 7, n = 14; P17, 89 ± 5, n = 14; P21, 104 ± 8, n = 10; P30, 82 ± 5, n = 11; KW ANOVA: p < 0.001; MW test: P14 vs. P17, p < 0.001; P14 vs. P21, p = 0.08; P14 vs. P30, p < 0.001; P17 vs. P21, p = 0.25; P17 vs. P30, p = 0.29; P21 vs. P30, p = 0.06; in L5: P14, 62 ± 5, n = 10; P17, 61 ± 3, n = 14; P21, 59 ± 5, n = 10; P30, 73 ± 5, n = 10; KW ANOVA: p = 0.1; in L6: P14, 134 ± 7, n = 13; P17, 105 ± 10; P21, 96 ± 5, n = 10; P30, 76 ± 3, n = 11; KW ANOVA: p < 0.001; MW test: P14 vs. P17, p = 0.005; P14 vs. P21, p = 0.001; P14 vs. P30, p < 0.001; P17 vs. P21, p = 0.8; P17 vs. P30, p = 0.004; P21 vs. P30, p < 0.001). Only p values <0.008 are considered significant owing to the correction for multiple comparisons). We then quantified frequency and amplitude of sEPSCs and sIPSCs across cortical layers between P14 and P30  rats and age-matched nondeprived littermates (Control P17). Thin, fixed slices obtained from the two groups of rats were immunostained with antibodies against PV and SST  in L2/3: Control, 79 ± 4, n = 11; bDEO P17, 100 ± 6, n = 13; unpaired t test, p = 0.01; in L4: Control, 89 ± 5, n = 14; bDEO P17, 114 ± 7, n = 13; unpaired t test, p = 0.006; in L5: Control, 61 ± 3, n = 14; bDEO P17, 59 ± 4, n = 12; unpaired t test, p = 0.1; in L6: Control, 105 ± 10, n = 13; bDEO P17, 125 ± 11, n = 14; unpaired t test, p = 0.2. Increased Rin is typically associated with an increase in the amplitude of synaptic responses, but we observed a reduction.A similar pattern of changes was observed when quantifying the effects of bDEO on inhibitory synaptic transmission. There was a significant decrease in inhibitory charge in L2/3, L4, and L5 after 3-d bDEO, but all parameters returned to baseline if eye opening was delayed by 7 d . Interesn = 11); bDEO P17, 0.46 ± 0.07 (n = 13); unpaired t test, p = 0.9. L4: Control P17, 0.54 ± 0.08 (n = 14); bDEO P17, 0.45 ± 0.05 (n = 13); unpaired t test, p = 0.4. L5: Control P17, 0.31 ± 0.06 (n = 14); bDEO P17, 0.37 ± 0.08 (n = 12); unpaired t test, p = 0.5. L6: Control P17, 0.46 ± 0.10 (n = 13); bDEO P17, 0.42 ± 0.06 (n = 14); unpaired t test, p = 0.7). No change in E/I ratio was observed for 7-d bDEO as well . The percentage change of the E/I ratio in all layers for bDEO P17 and bDEO P21 is represented in Excitatory and inhibitory charge were reduced proportionally in L2/3, L4, and L5; thus the E/I ratio was not affected by 3-d bDEO .Monocular DEO did not affect the number of PV opening D, E. Pern = 10; mDEO P17, 0.24 ± 0.04, n = 11; unpaired t test, p < 0.0003). In contrast, in L4, 3-d mDEO induced no change in excitatory transmission but a significant increase in sIPSC charge and average amplitude , distinct synaptic mechanisms underlie the shift in E/I balance in these two superficial layers. Excitatory and inhibitory synaptic transmission onto pyramidal neurons located in L5 and L6 were not affected by 3-d mDEO . These data indicate that synaptic drive onto pyramidal neurons in the deep layers of V1 is not sensitive to brief, unilateral delays in the onset of vision.Analysis of synaptic transmission, on the other hand, unveiled laminar-specific differences in the effects of mDEO. In L2/3, 3-d mDEO reduced excitatory charge through a decrease in sEPSC frequency, but no change in sEPSC average amplitude . No chanmplitude . Thus, w3-d mDEO A, B, as n = 14); mDEO P21, 0.27 ± 0.03 (n = 13); unpaired t test, p = 0.7).Interestingly, if eye opening was delayed unilaterally for 7 d (from P14 to P21), the decrease in excitatory charge in L2/3 persisted, although in this case it was not due to a decrease in frequency but rather to a decrease in average sEPSC amplitude . Furthern = 12); mDEO P21, 0.39 ± 0.04 (n = 11); unpaired t test, p = 0.4).In L4, the increase in inhibitory charge persisted if mDEO was maintained for 7 d, although the underlying mechanisms changed: at P21 there was a significant increase in sIPSC frequency, but no change in average sIPSC amplitude . In thisn = 14); mDEO P21, 0.40 ± 0.08 (n = 11); unpaired t test, p = 0.06). Excitatory and inhibitory synaptic drive onto L5 pyramidal neurons were not affected by mDEO of any duration, suggesting that synaptic transmission onto neurons in the output layer of V1 is not sensitive to unilateral manipulations of the time of eye opening ; mDEO P21, 0.15 ± 0.02 (n = 10); unpaired t test, p = 0.6). The percentage change in E/I ratio for mDEO P17 and mDEO P21 are reported in In L6, there was a significant increase in sEPSC charge and frequency, but no change in sEPSC amplitude after 7-d mDEO , while iThe layer-specific changes in E/I ratio induced by 3-d mDEO are represented in in (MΩ) in L2/3: P17 Control, 79 ± 5, n = 10; 3-d mDEO, 71 ± 2 (n = 11); unpaired t test, p = 0.6; P21 Control, 79 ± 5 (n = 14); 7-d mDEO, 80 ± 5 (n = 13); p = 0.9; in L4: P17 Control, 95 ± 6 (n = 11); 3-d mDEO, 106 ± 4 (n = 12); p = 0.2; P21 Control, 102 ± 12 (n = 11); 7-d mDEO, 87 ± 5 (n = 12); p = 0.3; in L5: P17 Control, 67 ± 6 (n = 9); 3-d mDEO, 70 ± 3 (n = 12); p = 0.7; P21 Control, 70 ± 10 (n = 10); 7-d mDEO, 72 ± 9 (n = 10); p = 0.9; in L6: P17 Control, 121 ± 9 (n = 11); 3-d mDEO, 136 ± 10 (n = 10); p = 0.25; P21 Control, 113 ± 7 (n = 14); 7-d mDEO, 115 ± 9 (n = 11); p = 0.8.All the effects reported above were not due to changes in input resistance, as this parameter was not affected by mDEO in any recorded neuron at either P17 or P21. ROur data indicate that mDEO has profound effects on V1 that not only are laminar specific, but also depend on the duration of the manipulation. Although the effect of 3-d mDEO was to shift the E/I balance toward inhibition in both L2/3 and L4, a longer manipulation restored the E/I ratio to control levels in both layers. The readjustment of the E/I balance was not dependent on the restoration of synaptic transmission to control levels, but on the recruitment of additional synaptic changes.Taken together, our results demonstrate that bDEO and mDEO have profoundly different effects on V1 circuitry. Whereas bDEO alters synaptic transmission while maintaining E/I balance and coordination across layers, mDEO disrupts the coordination of superficial and deep layers and significantly affects the excitability of local circuits by shifting the E/I ratio. Altogether, our results suggest that shifts in the time of eye opening trigger multistep cascades of events that involve laminar-specific mechanisms for synaptic plasticity.+ neurons, as well as plasticity of synaptic transmission onto pyramidal neurons. Before eye opening, excitatory and inhibitory drive onto pyramidal neurons in primary thalamorecipient layers (L4 and L6) are excitation shifted or balanced. From the time of eye opening to the peak of the critical period, the E/I ratio shifts toward similar levels of dominant inhibition through the maturation of synaptic transmission , whereas the previous study did not specify the location under analysis. The increase in number of PV+ neurons in V1 is likely dependent on a developmental regulation in the expression of PV, as fate-mapping studies report that the migration of neurons from the medial ganglionic eminence, the primary source of inhibitory neurons to cortex, is complete by eye opening  and monocular delayed eye opening (mDEO) did not affect the percentage of PVentation , dark reentation . In addicross V1  or the ccross V1 . Finallycross V1 . In our Synaptic transmission onto pyramidal neurons was strongly modulated by the time of eye opening. Binocular or monocular eye opening delays of different durations were instrumental for revealing the capacity of V1 to engage distinct, laminar-specific mechanisms for experience-dependent rewiring. A 3-d binocular delayed eye opening reduced excitatory and inhibitory transmission with similar trends across V1 layers, apart from L6. The effects of this manipulation were fully compensated for when eye opening was delayed by 7 d. These results are consistent with previous work showing that delays in the onset of vision can delay physiologic plasticity in V1 , and witOn the other hand, unilateral delays in eye opening had laminar-specific effects. The impact of 3-d mDEO was significant not only on synaptic transmission, but also on the E/I ratio of charges onto pyramidal neurons in the superficial layers of V1, and on the coordination of synaptic changes across layers. Although increasing the duration of mDEO to 7 d recovered the E/I balance onto pyramidal neurons in all layers, changes in synaptic transmission persisted. These results suggest that binocular visual drive at the time of eye opening is necessary to preserve coordination across layers. After an initial shift in E/I balance in the superficial layers, additional plastic changes were triggered to compensate for the shift in E/I ratio, although overall synaptic transmission did not return to control. In L2/3, the decrease in excitatory drive induced by 3-d mDEO was compensated for by a decrease in inhibitory drive observed with 7-d mDEO, consistent with the recruitment of a scaling-down mechanism for inhibition . In contTaken together, our data indicate that the effects of mDEO onto V1 pyramidal neurons are laminar specific and long-lasting, likely engaging mechanisms distinct from those recruited by the short-lasting effects of bDEO. Our results demonstrate that coordination of developmental patterns of maturation can be disrupted, especially if the visual input to the eyes is unbalanced.Delayed eye opening early in life is known to have dramatic effects on the organization of the visual system. Children with congenital binocular or monocular cataracts show visual impairments that can have lasting consequences even after corrective surgery . StudiesThe consequences of delayed eye opening extend beyond altered receptive field properties, however, affecting sensitivity to high spatial frequencies and sensitivity to feature spacing that alter the perception of motion . MonoculThe different patterns of experience-dependent reorganization of laminar circuits we report for mDEO and bDEO suggest that distinct synaptic and circuit mechanisms may be involved in the development of different aspects of healthy visual function. One may speculate that the ability of V1 neurons to recruit compensatory mechanisms to recover activity after long-lasting binocular deprivation may facilitate recovery of V1 neuron response properties if the eyes are reopened. On the contrary, the reorganization of local circuits after monocular delayed eye opening may induce long-lasting changes in V1 neurons response properties, as synaptic changes persist even after the E/I balance has recovered to control levels. The results reported in our study support the idea that healthy visual function relies on the coordinated development of synaptic activity in all layers of V1. Disruption of such coordination may be the mechanism leading to persistent impairment in visual function even after incoming visual drive is restored.


P<0.00001). An ACD4C<343 × 106/l had a significant negative impact on the 5-year progression-free survival and the overall survival as compared with an ACD4C⩾343 × 106/l =66.7–79.5) versus 50.3% (95% CI=39.0–60.6), P<0.00001 and 83.3% (95% CI=77.1–88.0) versus 59.0% (95% CI=47.9–68.5), P<0.00000001, respectively). Multivariate analysis revealed that the ACD4C was an independent prognostic marker (hazard ratio=2.2 (95% CI=1.3–3.7), P<0.01). In conclusion, a low ACD4C at diagnosis served as an independent poor prognostic marker in patients with DLBCL.The absolute peripheral blood lymphocyte count at diagnosis is known to be a strong prognostic factor in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but it remains unclear as to which peripheral blood lymphocyte population is reflective of DLBCL prognosis. In this cohort, 355 patients with DLBCL treated with R-CHOP from 2006 to 2013 were analyzed. The low absolute CD4+ T-cell count (ACD4C) at diagnosis negatively correlated with the overall response rate and the complete response rate significantly ( An absolute lymphocyte count (ALC) is known to be a strong prognostic factor in DLBCL that is the most common non-Hodgkin lymphoma type. A low ALC, and high absolute monocyte count (AMC) in peripheral blood before and after R-CHOP therapy can predict short-term survival in DLBCL13 and a low ALC is also known to negatively affect survival, not in germinal center (GC)-type DLBCL but in non-GC-type according to Hans algorithm.14 Currently, tumor-specific CD4+ T cells are known to have a role in the immune response against cancer, because CD4+ T cells facilitate the activation of cytotoxic CD8+ T cells and the antitumor effects of CD8+ T cells are limited in the absence of CD4+ T cells.16 In addition, CD4+ T cells can also target cancer cells in the absence of CD8+ T cells.18 Abnormalities in the CD4+ T-cell compartment, including the Th1 and Th2 subsets and related cytokines, have been shown to affect the occurrence of non-Hodgkin lymphoma, demonstrating that CD4+ T cells exert the strongest immunological response against tumors.20 On the other hand, it is not known whether peripheral blood T-cell subsets interfere with the prognosis of DLBCL. Recently, the ACD4C was identified as a prognostic marker in indolent lymphomas.22 Even in DLBCL, ACD4C was suggested as a prognostic marker in patients with R-CHOP.23 To further investigate the relationships of the host immunity at diagnosis with survival of DLBCL patients after R-CHOP and with other prognostic factors of DLBCL are crucial factors for improving the standard therapy in DLBCL. Thus, we primarily investigated the relationships of the pretreatment absolute lymphocyte subset counts with survival and other prognostic factors in de novo DLBCL patients.Rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) therapy is indicated for diffuse large B-cell lymphoma (DLBCL) patients regardless of the revised International Prognostic Index (IPI).de novo DLBCL by expert hematopathologists according to the World Health Organization classification,24 at a single cancer institute between 2006 and 2013. The cell of origin of DLBCL was assessed using the Hans algorithm.25 Patients were excluded if they had DLBCL secondary to non-Hodgkin lymphoma, clinically relevant heart disease or antibodies against human immunodeficiency virus. All patients provided informed consent to receive treatment and written informed consent allowing the use of their medical records. The review boards at our institute approved a retrospective review of medical records, which was conducted in accordance with the Declaration of Helsinki. Patients were staged according to the Ann Arbor classification.26 All patients underwent a physical examination, lymph node biopsy and bone marrow aspiration and biopsy. The disease stage was assessed using enhanced computerized tomography scans from the neck to the pelvis and whole-body positron emission tomography scans. The following clinical and laboratory data were available at the time of diagnosis: age, gender, ALC, absolute B-cell count, ACD4C, absolute CD8+ T-cell count (ACD8C), AMC, serum lactate dehydrogenase (LDH), serum albumin, performance status (PS), B symptoms, clinical stage, β2-microglobulin level, soluble IL-2 receptor and bone marrow involvement. Lymphocyte subset counts were calculated from the percentages obtained by flow cytometry.27 All data were collected and recorded in a computerized database.All patients included in this study were older than 18 years and they were histologically diagnosed with untreated 28 Responses were determined according to the International Workshop criteria.29All patients were scheduled to receive six to eight full cycles of R-CHOP-21 therapy.P-value was<0.05. EZR v3 was used for the statistical analysis.30Survival endpoints were evaluated using the Kaplan–Meier method and Cox proportional hazard model. Optimal cutoff values of other lymphocyte populations at diagnosis were determined on the basis of the utility of these parameters as markers of survival using receiver operating characteristic (ROC) curves. Fisher's exact test was used to determine the relationships between the absolute number of lymphocyte subsets and the prognostic factors of DLBCL evaluated as nominal variables. Pearson's correlation coefficients were used to evaluate the association between the absolute number of lymphocyte subsets and the continuous variables known as prognostic factors of DLBCL. Differences between the results of comparative tests were considered significant if the two-sided de novo DLBCL were included in the analysis (6/l (range 100–7080 × 106/l), 459 × 106/l (range 11–1944 × 106/l), 338 × 106/l (range 3–1518 × 106/l) and 372 × 106/l (range 0–5065 × 106/l), respectively. Regarding lymphocyte populations, 198 patients (56%) had a low ALC (<1380 × 106/l), 113 (32%) had a low ACD4C (<343 × 106/l), 68 (19%) had a low ACD8C (<191 × 106/l) and 107 (30%) had a high AMC (<529 × 106/l). GC DLBCL was diagnosed in 167 patients (53%), and non-GC DLBCL was observed in 148 patients (47%). Additionally, 26 patients (7%) received addition of radiotherapy after R-CHOP.A total of 414 patients were diagnosed with DLBCL at our institute from 2006 to 2013. Three hundred and fifty-five patients with analysis . The medThe overall response rate was 95% in the low IPI group (score=0–2) and 88% in the high IPI group (score=3–5). After a median follow-up of 57 months, the 5-year progression-free survival (PFS) and OS rates in the low IPI group were 76.0% and 83.7%, respectively, whereas those in the high IPI group were 41.8% and 55.4%, respectively. The 5-year PFS and OS in the GC group were 74.4% and 81.8%, respectively, whereas the corresponding rates in the non-GC group were 57.4% and 69.9%, respectively.6/l  among surviving patients versus 375 × 106/l  among patients who died (P<0.00001)). The absolute B-cell count before R-CHOP did not differ between surviving and deceased patients according to a box plot analysis, and no further evaluation was performed. Second, to determine the cutoff value for evaluating individual lymphocyte subsets at diagnosis, we performed area under the ROC curve analysis and then the optimal ALC, ACD8C and AMC cutoff values were determined as 1380 × 106, 191 × 106 and 549 × 106/l as negative survival markers, respectively (data not shown). In terms of ACD4C, ROC curve showed 343 × 106/l as a cutoff value  but also with ACD8C  using Pearson's correlation coefficients.First, box plot analysis illustrated that patients survived till the last follow-up after R-CHOP treatment had a higher ALC, ACD4C, ACD8C and AMC than those who died during the study period (blot plot of ACD4C is shown in P<0.001), 97% in the high ACD4C group versus 74% in the low ACD4C group (P<0.00000001) and 94% in the high ACD8C versus 72% in the low ACD8C (P<0.00001), whereas the overall response rate was not dependent on AMC . Similar trends were observed in the achievement of the complete response rate for the ALC  and ACD4C , and ACD8C , whereas the complete response rate was not dependent on AMC . The estimated PFS and OS rates were significantly different between high and low lymphocyte subsets. Kaplan–Meier analyses yielded the 5-year PFS rates of 76.5% =68.0–82.9) and 57.8% (95% CI=49.3–65.5) in the high and low ALC groups (P<0.0001), 73.7% (95% CI=66.7–79.5) and 50.3% (95% CI=39.0–60.6) in the high and low ACD4C groups (P<0.00001), 71.0% (95% CI=64.5–76.6) and 46.1% (95% CI=31.3–59.7) in the high and low ACD8C groups (P<0.00001), and 56.9% (95% CI=63.0–76.0) and 70.1% (95% CI=45.2–67.0) in the high and low AMC groups (P=0.01), respectively. Furthermore, the 5-year OS rates were 85.8% (95% CI=78.4–90.8) and 67.1% (95% CI=58.9–74.1) in the high and low ALC groups (P<0.0001), 83.3% (95% CI=77.1–88.0) and 59.0% (95% CI=47.9–68.5) in the high and low ACD4C groups (P<0.00000001), 80.2% (95% CI=74.4–84.8) and 55.6% (95% CI=40.1–68.7) in the high and low ACD8C groups (P<0.00001), and 68.8% (95% CI=72.0–83.5) and 78.4% (95% CI=57.4–77.7) in the high and low AMC groups (P=0.07), respectively.The overall response rate was higher in patients with high ALCs than those with low ALCs. The overall response rate was 96% in the high ALC group versus 84% in the low ALC group =2.2, 95% CI=1.3–3.7, P<0.01, 6/l at diagnosis was associated with an adjusted HR for OS after R-CHOP of 2.3 , non-GC type, low ACD4C, low ACD8C and high AMC. ACD4C<350 × 1031 β2-microglobulin and albumin using Pearson's correlation coefficients  or stage IV . The mean ACD4C values differed significantly between stage III and stage IV (P=0.04), P<0.001), P=0.03), R-value was 0.22, We identified correlations between ACD4C and known prognostic markers such as LDH, sIL-2R,P<0.00001), respectively. The OS was dependent on ACD4C in either the low or high IPI group . The 5-year OS rates were 84.7% (95% CI=75.8–90.5), 79.4% (95% CI=68.1–87.1), 76.2% (95% CI=61.4–85.9) and 48.9% (95% CI=32.2–63.7) in the high ACD4C/GC, high ACD4C/non-GC, low ACD4C/GC and low ACD4C/non-GC groups (P<0.000001), respectively. The OS differed with respect to the ACD4C significantly not only among patients with GC DLBCL but also among patients with non-GC DLBCL. The OS was significantly lower in the low ACD4C group (P=0.04 for GC type and P<0.000001 for non-GC type). The 5-year OS rates were 83.4% (95% CI=76.2–88.6), 83.0% (95% CI=68.3–91.3), 65.1% (95% CI=50.9–76.2) and 48.3% (95% CI=30.8–63.8) in the high ACD4C/low AMC, high ACD4C/high AMC, low ACD4C/low AMC and low ACD4C/high AMC groups (P<0.0000001), respectively. OS was significantly lower in the low ACD4C group, despite AMC was low (P<0.001) or high (P<0.001), compared with the high ACD4C group. High AMC tended to affect survival in the low ACD4C (P=0.07), but high AMC did not affect OS in the high ACD4C group in this cohort (P=0.94). As mentioned above, the OS was synergistically lower in the low ACD4C group among patients with high IPI, non-GC DLBCL or high AMC, respectively.We investigated the influence of ACD4C at diagnosis combined with IPI, cell-of-origin or AMC for DLBCL patients. Kaplan–Meier curves revealed that low ACD4C showed its influence in survival only in DLBCL patients with high IPI or non-GC. A low ACD4C affected survival outcomes in both the low and high IPI groups. The 5-year OS rates were 86.9% (95% CI=80.2–91.5), 73.9% (95% CI=58.9–84.1), 70.9% (95% CI=54.6–82.1) and 37.2% (95% CI=21.9–52.6) in the high ACD4C/low IPI, lowACD4C/low IPI, high ACD4C/high IPI and low ACD4C/high IPI groups  and 58.7% (95% CI=46.0–69.3) in the high CD4MR and low CD4MR groups (P<0.00001), respectively.We further investigated the influence of the ACD4C to AMC ratio (CD4MR) for DLBCL patients, because lymphocyte to monocyte ratio is known as a prognostic marker in DLBCL.6/l had a significant negative impact on the 5-year PFS and OS as compared with ACD4C⩾343 × 106/l. Low ACD8C and high AMC were not independent prognostic factors in this cohort. As ACD4C and ACD8C were strongly correlated with ALC, we excluded ALC from multivariate analysis. A low ACD4C combined with high IPI, non-GC type or high AMC aggravated the survival synergistically, suggesting the importance of synergistic worsening of prognosis due to the influence of these combined factors. Furthermore, low CD4MR affected negative prognosis significantly in DLBCL patients. A cutoff value for the ACD4C was chosen on the basis ROC curve. The initiation of antiretroviral therapy at the level of ACD4C<350 × 106/l is beneficial for human immunodeficiency virus-infected patients, which results in a significant decline in the risk of AIDS-related morbidity and mortality.38 These data suggest that ACD4C 340–350 × 106/l is crucial value for human immunity against infection or tumor. Recently, other group measured peripheral ACD4C in 43 DLBCL patients.24 In the study, 34 patients (72%) were given R-CHOP and mean value of ACD4C (460 × 106/l) was used as a cutoff value. They documented that low ACD4C was associated with poor PFS using the whole population, whereas ACD4C had an insignificant association with survival in the population treated with rituximab in multivariate analysis, owing to the small numbers. The presumption of Judd's study was that ACD4C predicted survival was validated in the present study. In addition, ALC, ACD8C and natural killer cell counts were insignificantly associated with PFS and OS in the study,23 which supports the results of our study.In the present study, we aimed to identify which lymphocyte subsets are most associated with therapeutic outcomes in patients with DLBCL who were treated with R-CHOP. Multivariate analysis illustrated that ACD4C<343 × 10et al. and Zhang et al. described that a low ACD4C was not associated with poor-prognostic markers in patients with follicular lymphoma21 and mantle cell lymphoma.22 By contrast, a low ACD4C at diagnosis exhibit significant inverse correlations with the prognostic factors in DLBCL, suggesting that an ACD4C correlates with poor prognostic factors only in aggressive B-cell lymphoma.We further identified the previously unclear relationships of advanced clinical stage, high LDH, low albumin, high β2-microglobulin, ⩾2 sites of extranodal disease and high sIL-2R with a low ACD4C. He Retrospective nature is a potential weakness of our study. Thus, further prospective investigations are required in the future, to determine whether the pretreatment ACD4C affects the survival in high-risk DLBCL patients treated with R-CHOP and whether low ACD4C combined with high IPI, non-GC type and high AMC aggravated the survival synergistically.de novo DLBCL, who were treated with R-CHOP. A low ACD4C combined with high IPI, non-GC type or high AMC aggravated the survival synergistically in DLBCL.In conclusion, we demonstrated that pretreatment peripheral ACD4C put a negative impact on survival in DLBCL patients, and that various poor prognostic factors of non-Hodgkin lymphoma were associated with a significant reduction in ACD4C in DLBCL. A low ACD4C at diagnosis had a significant negative impact on the survival of patients with


P<0.05), at T1-4-6-8 h for PS (P<0.05) and at T4-6-8 h for SCL (P < 0.0001). Regarding the mean intra-group values observed pre (T0) and post-surgery (from T15 min to T72 h), significant difference between the groups were found for RR (P<0.01), PS and SCL (P<0.05). The pharmacokinetic profile was best fitted by a two-compartmental model and characterized by a fast distribution half-life and slow elimination half-life  and meloxicam mean concentrations at 96 h was of 0.18 ± 0.14 μg/mL. The volume of distribution and clearance values were quite low, but reasonably homogenous among individuals . The IV administration of meloxicam in buffalo calves shows encouraging effects represented by significant and prolonged analgesic effects, significant reduction of SCL as well as similar pharmacokinetic profile to bovine calves.The aims of the investigation were to establish for the first time (i) clinical efficacy and (ii) pharmacokinetic profile of meloxicam intravenously (IV) administered in male Mediterranean buffalo calves after surgical orchiectomy. The study was performed on 10 healthy buffalo calves, between 4 and 5 months old and between 127 and 135 kg of body weight (b.w.). An IV injection of 0.5 mg/kg b.w. of meloxicam was administered in six calves  immediately after surgery; the other four animals were used as untreated control group (CG). The clinical efficacy of meloxicam was evaluated pre- and post-surgery by monitoring respiratory rate (RR), heart rate (HR), rectal temperature (T°C), serum cortisol levels (SCL) and pain score (PS). Significant inter-groups differences were detected at sampling times (T): 4 hour (h) for RR ( Bubalus bubalis) counts of approximately 395.000 heads  than CG (23.7 ± 6.4 bpm). Comparing the two groups at each sampling time, TG showed significant lower values than CG at T4 h after surgery .The overall mean RR values recorded were significantly lower (P < 0.01) than CG (T0 = 31.0 ± 9.2 bpm vs. mean T15 min-T72 h = 23.1 ± 5.9 bpm). No inter-  or intra-groups significant differences were instead recorded for HR and T°C.Instead, the mean intra-group values observed pre- (T0) and post-surgery (average from T15 min to T72 h) revealed significant lower values within TG . The mean overall pain scores recorded were significantly lower (P < 0.05) in TG (3.5 ± 3.3 points) than CG (4.9 ± 3.5 points). Comparing the two groups at each sampling time, TG showed significant lower values than CG at T1h , T4h , T6h  and T8h  after surgery. A significant difference between the mean intra-group values observed pre- (T0) and post-surgery (average from T15 min to T72 h) were detected both within TG  and CG .Regarding the PS, a near perfect agreement between the observers was found  in TG (26.8 ± 22.6 nmol/L) than CG (34.1 ± 18.6 nmol/L). Comparing the two groups at each sampling time, TG showed significant lower values than CG at T4h , T6h  and T8h , than in TG (T0 = 24.6 ± 9.2 vs. mean T15 min-T72 h = 27.0 ± 23.3 nmol/L).The overall mean SCL values recorded were significantly lower  and CG ; instead it was moderate between SCL and T°C in CG .Finally, a very high correlation was detected between SCL and PS both in TG , meloxicam administration can significantly reduce pain, stress and increase profitability by increasing body average daily gain and reducing susceptibility to disease ,21. MeloPain assessment in ruminants represents a true challenge for clinicians in field. Typically, prey in their natural state, they tend not to express pain to limit vulnerability . AlthougUnlike a similar study based on intravenous administration of meloxicam in dairy calves during dehorning , the HR Regarding the body temperature, the administration of meloxicam did not produce any significant difference between the two groups confirming similar finding recently observed in beef bull calves submitted to surgical castration . The corFinally, the overall significant difference observed between TG and CG regarding the PS seemed to confirm the painkiller properties of the NSAIDs allowing a reduction of pain behaviours. Although the surgical procedure produced a series of behavioural changes in both groups, treated animals were also scored significantly lower than the untreated, when compared with their respective T0 (pre-surgery) further supporting the hypothesis. Moreover, the significant correlations observed between PS and SCL in both the groups may confirm the reliability of this composite pain scale for assessing postoperative pain also in buffaloes.Concerning the SCL, the parameter showed lower overall mean values in TG than CG, as observed also by Coetzee at al.  in animamax) of 3.10 μg/mL (range 2.64–3.79 μg/mL) was recorded at 11.64 h (range 10–12 h) with an elimination half-life of 27.54 h (range 19.97–43.29 h) after oral meloxicam administration at 1 mg/kg. After IV administration at 0.5 mg/kg the elimination half-life and ClB were 20.35 h (range 17.84–22.76 h) and 6 mL/kg/h (range 4.8–7.2 mL/kg/h), respectively, whereas, Vdss and MRT were 171 mL/kg (range 151–189 mL/kg) and 28.19 (range 24.65–31.58), respectively [The pharmacokinetics of meloxicam after oral and IV administration has been first described by Coetzee at al. 2009  in calvedss and ClB values were reasonably homogenous among individuals, with values of 142.31 ± 55.08 mL/kg and 4.38 ± 0.95 mL/kg/h, respectively. As reported for cows, the high protein binding of meloxicam (96.5–98%) [The pharmacokinetic profile of meloxicam in buffalo was characterized by a fast distribution half-life (0.09 ± 0.06 h) and slow elimination half-life (21.51 ± 6.4 h) and after a single IV injection meloxicam was quantifiable for several days with a mean concentrations of 0.18 ± 0.14 μg/mL at 96 h. Despite the young age of our buffaloes and the presumable variable metabolic capacity, the low V6.5–98%)  can just6.5–98%) .B, is responsible for the quite long elimination half-life and is influenced by the hepatic metabolism, that is necessary for meloxicam elimination from the body [dss (142.31 ± 55.08 mL/kg) was very similar to the Vz (146.16 ± 53.98 mL/kg). Thus, in our buffaloes minimal amounts of meloxicam were eliminated during the distribution phase.The low Clthe body . The VdsB was more rapid in Holstein calves (6.64 ± 0.76 vs. 4.38 ± 0.95 mL/h/kg), but elimination half-lives and MRT were comparable . Volumes of distribution were higher in Holstein calves , whereas AUC was higher in buffaloes (118.81 ± 27.57 vs. 81.08 ± 10.58 h* μg/kg). The results in buffaloes were comparable also to those first published calves after IV administration at the same dose by Coetzee et al. 2009 [Bubalus bubalis) and cattle (Bos Taurus) can have a significant impact on pharmacokinetics [The pharmacokinetic profile observed in buffaloes was quite similar to that observed more recently in Holstein calves undergoing dehorning by Coetzee et al.  after IVkinetics ,53 was n50) of meloxicam in cattle, whereas in horse and dog meloxicam EC50 was reported of approximately 0.2 and 0.36 μg/kg, respectively [The association of the positive effects as painkiller together with the pharmacokinetic profile of meloxicam indicate that this drug can have effects for several days in buffalo calves. In our study, meloxicam was administered immediately after surgery, but to optimize the perioperative analgesia and in agreement with a pre-empitive analgesia, meloxicam could be administered IV to buffaloes 20–30 min before surgery. No studies have been published on effective plasma concentration Click here for additional data file.