Abundant nanopores in the conductive MOFs expose additional catalytic active sites, and the advantageous 2D π-conjugated structure helps accelerate charge transfer. Owing to the introduction of Rh, [Ni2.85Rh0.15(HHTP)2]n/CC exhibited substantially improved oxygen evolution reaction (OER) activity and exhibited only an overpotential of 320 mV to achieve the current density of 20 mA cm-2. The remarkable OER performance confirmed by the electrochemical tests could be ascribed to the synergistic effect caused by the doped Rh together with Ni in [Ni2.85Rh0.15(HHTP)2]n/CC, thereby exhibiting outstanding electrocatalytic performance.Metal–organic frameworks (MOFs) have recently been considered the promising catalysts due to their merits of abundant metal sites, versatile coordination groups, and tunable porous structure. However, low electronic conductivity of most MOFs obstructs their direct application in electrocatalysis. In this work, we fabricate an Ni–Rh bimetallic conductive MOF ([Ni Therefore, the kinetics are extremely sluggish, and large overpotentials are required to activate the reaction. In this case, high-activity electrocatalysts are required to overcome reaction barriers , a special type of crystalline porous materials have attracted immense attention for catalysis, owing to their enormous specific surface area and tunable pore dimensions . While txRhy)3(HHTP)2]n/CC using a simple one-step solvothermal method. Since the conductive MOFs were in situ securely grown on carbon cloth, the conductivity was greatly improved, and the electrochemical impedance was substantially reduced (xRhy)3(HHTP)2]n/CC was highly dependent on the Ni/Rh molar ratio. The optimized [Ni2.85Rh0.15(HHTP)2]n/CC showed the highest OER activity, with a low overpotential of 320 mV at the current density of 20 mA cm-2, similar to that of commercial RuO2/CC. The synergistic effect of Rh and Ni in [Ni2.85Rh0.15(HHTP)2]n/CC efficiently modulated the electronic state distribution of the two central metal atoms, hence altering the adsorption characteristics of oxygenous intermediates and improving the electrocatalytic activity and stability of OER.2,3,6,7,10,11-Hexahydroxytriphenylene (HHTP) is a typical triphenylene-based organic ligand, with an abundance of functional -OH groups, which can be coordinated with metal atoms to form 2D planar π-conjugated MOFs. Due to the charge delocalization and orbital overlap between the metal node and organic linker, this kind of MOFs are characterized with intrinsic conductivity and can be directly used as the electrocatalyst without pyrolysis activation. Herein, we have designed and synthesized Ni–Rh-based bimetallic conductive MOFs n/CC: Cobalt acetate (Ni(OAc)2·4H2O, 13 mg) and hexahydroxytriphenylene  were dissolved in 1.8 mL solution , followed by sonicating for 30 min. Afterward, the washed carbon cloth was put into the aforementioned solution. The reaction mixture was sealed into an autoclave, heated to 85°C, and maintained at this temperature for 12 h. After the autoclave was cooled down, the obtained carbon cloth was treated with deionized water and then freeze-dried for 24 h.xRhy(HHTP)2]n/CC and [NixRhy(HHTP)2]n powderSynthesis of [Ni: For the synthesis of [NixRhy(HHTP)2]/CC, 0.7 mg/mL of rhodium acetate [Rh(OAc)2] solution was prepared ahead of time and mixed into Ni(OAc)2 and HHTP solution, according to the set ratio. Three different ratios of Ni/Rh were considered, the dosages of which are summarized in xRhy(HHTP)2]n powders were similar to those for [NixRhy(HHTP)2]n/CC except without carbon cloth.Preparation of RuO2/CC: The RuO2 dispersion solution was prepared by adding 20 μL of 5% Nafion solution and 4 mg RuO2 into 780 μL of mixed solution . Next, the RuO2 dispersion solution was drop-coated onto a 0.5 cm × 2 cm carbon cloth. After this step, the obtained carbon cloth with set RuO2 loading was dried overnight at 60°C.Measurements: The phase composition of the sample was characterized by X-ray powder diffraction (XRD) using a Shimadzu XRD-6100 . The morphologies of samples were observed by transmission electron microscopy , and scanning electron microscopy (SEM) depictions were obtained using a Zeiss_Supra55 electron microscope. Fourier-transform infrared spectroscopy (FT-IR) was conducted in the range of 4,000–400 cm-1 on a Thermo Nicolet iS10 spectrometer. JEOL JEM 2100F was used for HRTEM and mapping. Raman spectra were recorded using HORIBA HR800 series.Electrochemical characterizations: Electrochemical measurements were investigated using the electrochemical potentiostat  in 0.1 M KOH at room temperature. The carbon cloth with the in situ grown catalyst was used as the working electrode directly. In addition, the platinum wire and the saturated calomel electrode were used as the counter and reference electrodes, respectively. All polarization curves were performed by linear voltammetric scan at a sweep rate of 5 mV s-1 and were iR-corrected. All potentials can be converted concerning the reversible hydrogen electrode (RHE), based on the following Nernst equation: E(vs. RHE) = ERef + 0.059*13 + ETest. The electrochemical impedance spectroscopy (EIS) test was conducted on the working electrodes with an overpotential of 0.2 V, a frequency range of 0.01 ∼ 105 Hz, and an amplitude of 5.0 mV.xRhy)3(HHTP)2]n could be properly designed and synthesized with the one-step solvothermal method, which is schematically demonstrated in in situ growth process efficiently bonded these [NixRhy(HHTP)2]n MOFs to the backbone of the carbon cloth, they can be directly used as the OER electrodes. Differing from the smooth surface of the original carbon cloth (3(HHTP)2]n/CC displayed a homogeneous distribution of regular nanorods (2.85Rh0.15(HHTP)2]n/CC had an irregular structure, suggesting that the doping of Rh had disrupted the process of crystallization and changed the rod-like morphology. As shown in 2.85Rh0.15(HHTP)2]n/CC surfaces 2]n/CC and [Ni3(HHTP)2]n/CC samples, as shown in -1, which corresponded to the D and G peaks of the carbon material, respectively. The ID/IG ratios obtained for [Ni2.85Rh0.15(HHTP)2]n/CC and [Ni3(HHTP)2]n/CC were 0.756 and 0.77, respectively, indicating that both [Ni2.85Rh0.15(HHTP)2]n/CC and [Ni3(HHTP)2]n/CC displayed the similar graphitized structure. This is because the graphitized structure is mainly ascribed to the carbon cloth, and the solvothermal reaction did not influence the property of the carbon cloth.XRD was used to determine the phase and structure of [Ni2.85Rh0.15(HHTP)2]n/CC (2.85Rh0.15(HHTP)2]n/CC can be fitted into two characteristic peaks located at 284.6 eV and 286 eV, corresponding to C-O and C=C species, respectively (1/2 and Ni(II) 2p3/2, respectively, indicating the existence of Ni with a single valence state 2]n/CC sample has pure phase composition, without the formation of other heterogeneous phases (X-ray photoelectron spectroscopy (XPS) was adopted to investigate the surface chemistry and valence state within the prepared samples. The survey spectra of [Ni2]n/CC  displayeectively . Additioectively , indicatectively . The higectively  demonstrce state . The broce state , the twoent form . In combs phases .3(HHTP)2]n/CC and therefore accelerate the OER process. However, excessive doping of Rh resulted in decreased OER activity, which was ascribed to the structure collapse and consequent conductivity attenuation of the conductive MOFs. [Ni2.85Rh0.15(HHTP)2]n/CC displayed the lowest potential of 1.55 V at 20 mA cm−2 compared to its single-metal counterpart (1.63 V for [Ni3(HHTP)2]n/CC) or other bimetallic conductive MOFs with different metal ratios (1.61 V for [Ni2.94Rh0.06(HHTP)2]n/CC and 1.6 V for [Ni2.7Rh0.3(HHTP)2]n/CC). It is noteworthy that the additional peak of 1.4 V, as shown in 2+. It was agreeable to see in 2.85Rh0.15(HHTP)2]n/CC displayed an excellent OER activity, even similar to that of RuO2/CC in terms of the potential at 20 mA cm−2, under the same mass loading of 0.825 mg. In addition, there was very poor electrocatalytic activity on the blank carbon cloth, implying that the excellent electrocatalytic performance was derived only from conductive bimetallic MOFs [Ni2.85Rh0.15(HHTP)2]n/CC (3(HHTP)2]n/CC, [Ni2.94Rh0.06(HHTP)2]n/CC, [Ni2.85Rh0.15(HHTP)2]n/CC, [Ni2.7Rh0.3(HHTP)2]n/CC, and RuO2/CC were 120, 82.9, 65.6, 64, and 140 mV dec−1, respectively. This result indicated that the bimetallic MOFs achieved faster reaction kinetics than the commercial RuO2 catalysts. In order to explore the charge transfer capability at the interface of the catalyst/electrolyte, the EIS test was performed. The Nyquist plots for different catalysts are provided in 2.85Rh0.15(HHTP)2]n/CC, indicating the doped Rh in favor of charge transfer during the OER process. In addition to the activity, long-term stability was another key concern for the practical application of electrocatalysts. Therefore, a chronoamperometry (I-t plot) response was performed at the constant potential of 1.6 V in 0.1 M KOH solution to assess the OER stability. 2.85Rh0.15(HHTP)2]n/CC catalyst retained 86% of the initial current density after 45,000 s, which is larger than that of 79% for RuO2/CC, manifesting a better stability of [Ni2.85Rh0.15(HHTP)2]n/CC.The electrocatalytic activity of as-prepared conductive MOF electrocatalysts for OER was investigated in 0.1 M KOH solution using a typical three-electrode system. The linear sweep voltammetry (LSV) curves of the MOF samples with various Rh doping concentrations are shown in 2]n/CC . To furt2.85Rh0.15(HHTP)2]n/CC were successfully synthesized with a simple solvothermal method. The obtained conductive MOF showed prominent OER activity comparable to RuO2/CC and outperformed RuO2/CC in terms of long-term stability. The remarkable activity could be attributed to the abundant active metallic sites in the MOFs, which are in favor of reactant adsorption and activation. The porous morphology leads to high-efficient mass transfer and ion penetration. In addition, the conductive characteristics of the MOFs facilitate charge transfer during the OER. The multi-aspect merits synergistically improve the electrocatalytic activity. Therefore, this work provides a facile method to synthesize the bimetallic conductive MOFs, being directly used as the efficient OER electrocatalysts.In summary, the conductive MOFs [Ni


Aloe vera extract with antimicrobial, antitumor, antiviral, and antioxidant activities. This study aims to optimize the synthesis of acemannan from methacrylate powder using a simple method and characterize it for potential use as a wound-healing agent.Acemannan is an acetylated polysaccharide of 1H-nuclear magnetic resonance (NMR). 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assays were performed to investigate the antioxidant activity of acemannan and its effects on cell proliferation and oxidative stress damage, respectively. Further, a migration assay was conducted to determine the wound healing properties of acemannan.Acemannan was purified from methacrylated acemannan and characterized using high-performance liquid chromatography (HPLC), Fourier-transform infrared spectroscopy (FTIR), and A. vera, with the FTIR revealing peaks at 1739.94 cm−1 (C = O stretching vibration), 1370 cm−1 (deformation of the H-C–OH bonds), and 1370 cm−1 (C–O–C asymmetric stretching vibration); 1H NMR showed an acetylation degree of 1.202. The DPPH results showed the highest antioxidant activity of acemannan with a 45% radical clearance rate, compared to malvidin, CoQ10, and water. Moreover, 2000 µg/mL acemannan showed the most optimal concentration for inducing cell proliferation, while 5 µg/mL acemannan induced the highest cell migration after 3 h. In addition, MTT assay findings showed that after 24 h, acemannan treatment successfully recovered cell damage due to H2O2 pre-treatment.We successfully optimized the synthesis of acemannan from methacrylate powder using a simple method. Our results demonstrated that methacrylated acemannan was identified as a polysaccharide with an acetylation degree similar to that in Our study provides a suitable technique for effective acemannan production and presents acemannan as a potential agent for use in accelerating wound healing through its antioxidant properties, as well as cell proliferation- and migration-inducing activities. Arnebia euchroma hydrogel [Skin wound healing is a remarkably regulated cellular and molecular mechanism that directly begins after tissue disruption until wound closure . In peophydrogel , extracthydrogel , collagehydrogel , and alghydrogel  have beeAloe vera is a medicinal plant that has traditionally been used to treat a variety of diseases and skin lesions [A. vera extract displays antioxidant action, lowering intracellular ROS levels in H2O2 treated cells and activating antioxidant defense systems and wound healing via Nrf2 activation [in vitro and improved wound healing [A. vera gel not only increases the quantity of collagen production in wounds, but also changes the composition of collagen, promotes collagen cross-linking, thus advancing wound healing [A. vera such as acetylated acemannan [A. vera considerably differ based on their extraction method. Thunyakitpisal et al. reported that the fresh gel extracted from A. vera using water extraction and separation (Shodex Sugar KS-804 column) produced 0.04% monosaccharides with the composition Mannose: Glucose: Galactose (65:17:17) [A. vera to produce maximum acemannan yield. Therefore, this study aimed to develop a suitable method for acemannan production and its characterization for optimum yield and quality, with subsequent potential application in clinical settings, including wound care. lesions . A. verativation . These a healing . A. vera healing . Because healing . The polcemannan , have becemannan , metabolcemannan  diseasescemannan . Acemanncemannan , 20. Xincemannan . However5:17:17) , whereas5:17:17) . These dThe synthesis of methacrylated acemannan (MACE) was performed using powdered acemannan  obtained from Lorand Laboratories . The powder was dissolved in deionized water (2.5% w/v) at 50 °C and shaken for 8 h until the polymer was fully solubilized. Then, the solution was reacted with 8% methacrylic anhydride  and incubated for 6 h at 50 °C. The pH of this reaction mixture was maintained at 8.0 and regularly adjusted with 5.0 M NaOH solution in distilled water. The solution was then dialyzed against distilled water using 12–14 k-CA cut-off dialysis tubing  for 3 d at 25 °C to remove debris and unreacted MA. The solution was then frozen at –80 °C, lyophilized to form a powder, and purified in 85% ethanol for 48 h. The purified acemannan was frozen at –80 °C again and lyophilized at 0.08 mbar and –77.7 °C. The obtained powder was protected from light and stored at 4 °C until further use .The acemannan content was measured using high-performance liquid chromatography (HPLC). The powder was dissolved in a 0.5% acidic solution of sodium and shaken for 8 h. The acidic aqueous solution was filtered using a 0.22-μm filter. The acemannan content was detected using an HPLC  system composed of a TosoHaas column , equipped with a SIL-10A injector and UV lamp (Perkin Elmer 235C). Standard acemannan (0.2%) was used as the reference.−1 at 25 °C.Moreover, Fourier-transform infrared spectroscopy (FTIR) was performed to verify the chemical characteristic of acemannan. The samples were analyzed in a Nicolet iS50 FTIR spectrometer  using the attenuated total reflectance technique to obtain 32 scans with a resolution of 2 cm1H-Nuclear Magnetic Resonance (1H-NMR) spectroscopy was performed using VNMR-J 2.2 c on a Varian VNMRS-500 MHz spectrometer with a 1H-19F/15N-31P 5 mm PFG AutoX DB probe: relaxation delay, 10 s; number of scans, 32; sweep width, 16 ppm; point count, 32 768; pulse angle, 45°; and acquisition time, 2.049 s. All samples were loaded using an automated sample delivery system. For quantitative analysis, the spectra were processed with LB = 0.7 Hz, phase and baseline correction, and weight and Fourier transform function by ACD/Labs 10.0 software; the resonance signals were integrated manually.In addition, 2O was used as a control. Furthermore, 100 μL of each sample was mixed with 900 μL DPPH working solution, stored for 30 min to 24 h at 25 °C in the dark, and centrifuged , and the absorbance was measured at λ = 570 nm using a microplate reader .To prepare a stock solution, 19.7 mg of 2,2-diphenyl-1-picrylhydrazyl  was dissolved in 100 mL methanol and diluted at 1:4.55 to a working solution. The absorbance was measured at ~1.38, λ = 515 nm. Acemannan, malvidin, and CoQ10 were prepared at 45 µM, 54 µM, and 45 µM, respectively. H6 cells per dish. Approximately 48 h after inoculation, cultured RGM1 cells formed a confluent monolayer sheet.Normal mucus-producing epithelial cells of rat gastric mucosa (RGM1) were purchased from Bioresource Collection and Research Center, Taiwan. The cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12   and supplemented with 5% heat-inactivated fetal bovine serum . The cells were inoculated onto collagen-type I-coated plastic culture dishes (60-mm diameter) at a density of 5 × 102 atmosphere. The medium was renewed every 2–3 d.Cell culture of human fibroblast cells (CG1639), provided by the Bioresource Collection and Research Center, Taiwan, was performed in a T-75 flask  with high-glucose DMEM  containing 15% FBS  and 1% antibiotic-antimycotic  in an incubator at 37 °C and a 5% CO2 of available surface area. The medium was carefully removed after 7 d of culture and replaced with 50 mL of fresh DMEM medium for the subsequent 7 d.NCTC clone 929  were purchased from Bioresource Collection and Research Center, Taiwan. L929 cells were grown for three passages from cryogenic storage before seeding for secretion collection. The cells were seeded in 50 mL of high glucose DMEM containing 10% (vol/vol) FBS, 1 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at a density of ∼6,500 cells per cmMus musculus were obtained from Bioresource Collection and Research Center, Taiwan. The cells were grown in DMEM containing 4.5 g/L glucose, 10% (w/v) FBS, and 100 μg/mL penicillin and 0.1 mg/mL streptomycin (complete culture medium) at 37 °C in an incubator with a 5% CO2 atmosphere until confluence and ready for further usage.Furthermore, 3T3-L1 fibroblast cell lines from 2HPO4, and 1.76 mM KH2PO4, at pH 7.4). First, the cultured RGM1, L929, and CG1639 cells at a density of 1×104 cells/well were treated with different concentrations of acemannan  and incubated at 37 °C in an incubator humidified with 5% CO2 for 24 h. The medium from each cell group was then removed and replaced with MTT solution (10 μL per 100 μL medium) and incubated at 37 °C for 4 h. The supernatant was carefully removed and 100 mL dimethyl sulfoxide (DMSO) solution was added to lyse the cells for 10 min. The absorbance was detected using a microplate reader  at 570 nm and performed as three independent experiments.Cell proliferation was analyzed using 3--2,5-diphenyl-2H-tetrazolium bromide  powder dissolved to a final concentration of 5 mg/mL in 1 X phosphate-buffered saline (PBS)  in L929 cells. First, the L929 cells were seeded on 96-well culture plates  and cultured overnight at 37 °C in an incubator humidified with 5% CO2 until they reached a density of 1×105 cells/well. Subsequently, 0.8 mM H2O2 was added to the cultures and incubated for 30 min at 37 °C, followed by 9 µM of acemannan with incubation for another 30 min. After the treatment, MTT solution was added to each well, and the plates were incubated at 37 °C in an incubator humidified with 5% CO2 for 1 h. The supernatant was removed, and the cells were lysed completely in 100 mL DMSO. The absorbance was detected using a microplate reader  at 570 nm and performed as three independent experiments.We examined the antioxidant effect of acemannan against oxidative stress induced by hydrogen peroxide  were seeded and cultured in 24-well plates with DMEM culture medium and 10% FBS until they attained a density of 1×104 cells/well. The IBIDI culture inserts were removed, and the two cell islands were washed with PBS to remove debris. The culture plate was incubated overnight at 37 °C in an incubator humidified with 5% CO2. The cells were treated with DMEM with different concentrations of acemannan , while the positive control was treated with DMEM supplemented with 10% FBS and the negative control with 5% FBS. Images of the scratch area (wound area) at 0 h were taken using a built-in camera in the microscope (40 X magnification), and then the plate was incubated at 37 °C in an incubator humidified with 5% CO2 for 3, 6, and 9 h. Alterations in the injured area after different time points  were captured. Data were evaluated using ImageJ software version 1.53t 2022  to calculate the percent wound area.Culture-inserts  plated on 24-well plates  were used to perform the p < 0.05, **p < 0.01, and ***p < 0.001.All data were analyzed using the one-way analysis of variance or the two-tailed paired t-test of IBM® SPSS® Software Version 18.0 . Significant differences between groups were set at *A. vera are partially due to acetylated mannan (acemannan). This necessitates characterizing its chemical content to enhance knowledge of its physical, chemical, or biological functions [-1 (C=O stretching vibration), 1370 cm-1 (deformation of the H-C-OH bonds), and 1370 cm-1 (C–O–C asymmetric stretching vibration) -D-mannosyl residues acetylated at the C-2 and C-3 positions that display a mannose and galactose attached to C-6 [A. vera gel due to the inability of humans to enzymatically degrade these bonds after treatment [A. vera is structurally unique, making it a characteristic compound of Aloe species among other well-known mannans [In addition, cemannan , 30. Ourd to C-6 . This coreatment . The ace mannans .Fig. 21H2O  showed the highest absorbance compared with acemannan-H2O2 pretreatment. This suggests that acemannan treatment can increase cell viability in damaged or healthy cells, owing to its antioxidant effect [MTT assay was performed to assess the antioxidant activity of acemannan on cells after induction of cell damage  healing . H2O2 priability . In our rol Fig. . The rest effect .Fig. 5AnThe primary goal of epithelial cells in wound healing is to restore the broken epithelial barrier after injury . TherefoA. euchroma hydrogel on wounds [A. vera gel displayed considerable stimulatory effects on fibroblast and keratinocyte cell proliferation and migration, as well as strong protective benefits against preservative-induced keratinocyte death. Those abilities in plants are likely due to numerous element contents, including polyphenols, flavonoids, phenolic acids, and acemannan which are responsible for suppressing free radicals and antioxidant effects in plants [A. vera hydrogel specifically can enhance wound healing in vivo by promoting cell proliferation and differentiation [A similar study was also conducted by Tavakolizadeh et al., which demonstrated strong tissue adhesiveness (adhesive strength of up to 48 N m-1) and outstanding therapeutic qualities  of n wounds  used anon plants . Acemannntiation . This suntiation –51 prope2O2. We believe that our study provides a better technique for acemannan production, as well as shows the potential of acemannan in accelerating the wound healing process.In summary, we successfully optimized the synthesis of acemannan from methacrylate powder using a simple method and adequate characterization techniques. Our results suggest that acemannan is a polysaccharide that has high and stable antioxidant properties with 24-h clearance activity compared to CoQ10, malvidin, and water. Acemannan was also able to stimulate cell proliferation and migration, as well as promote cell recovery after damage caused by H


Therefore, increasing the inclusion level of supplementary duckweed up to 50% improves growth performance, carcass quality, and yield of Horro ram.The study was aimed at evaluating growth performance, carcass, and non-carcass traits of Horro rams fed on commercial feed (CO) supplemented with different proportions of duckweed (DW). For the study, twenty-four yearling Horro rams initially weighing on average 17.73 ± 0.30 kg were used with a 15-day adaptation period (none-study period) and a 90-day study period. Each animal was randomly assigned to three treatments; T1 (100% CO), T2 (25% DW + 75% CO), and T3 (50% DW + 50% CO) with two replications each containing four animals. All diets were isocaloric and isonitrogenous and formulated according to the nutrient requirements of sheep. The data were analyzed using SAS. Results indicated that dry matter intake in T3 (914.37 g. d-1) was higher (P < 0.001) than in T1 (849.12 g. d-1) and T2 (870.50 g. d-1). Average daily body weight gain in T3 (77 g. d-1) was higher (P < 0.001) than in T1 (48.60 g. d-1) and T2 (58.50 g. d-1). Feed conversion efficiency was higher (P < 0.001) for T3 (0.08) followed by T2 (0.07) and T1 (0.05). Final body weight (24.56 kg), empty body weight (20.40 kg), hot carcass weight (10.27 kg), dressing percentage (42.01) and rib-eye muscle area (9.11 cm  Though  species , its yie species .Duckweed grown under ideal conditions contains 17.50%–37.00% total protein on a dry weight basis , 59.33% 22.1The study was conducted at Ambo University, Guder Mamo Mezemir Campus, West Shoa Zone, Oromia Regional State, Ethiopia, located 124 km west of Addis Ababa. The study site is situated at 8°17′ N latitude and 37°1′ E longitude and at mid-altitude that ranges from 1380 to 3300 m above sea level. The mean annual rainfall of the area is 1079 mm, and the mean minimum and maximum daily temperatures of the area are 12 and 29 °C, respectively .2.22 on a dry matter basis has been harvested. Harvested duckweed was spread out in sunlight for partial drying at 30%  orally and ivermectin injection subcutaneously for internal parasites and sprayed with acarmic acaricide for external parasites early. After an adaptation period of 15 days (none-study period), the feeding trial lasted 90 days (study period).2.5The carcass was evaluated after overnight fasting and slaughtering the animals. To slaughter, the animals’ jugular vein and carotid artery were severed using a knife after which blood was weighed. After slaughter, the skin was flayed and weighed. All internal organs, heads, and feet were removed and weighed. To estimate the hot carcass weight, the weight of legs below the hook and knee joints, thorax, head, abdominal, and pelvic cavity contents were removed.2) was measured at the 12th and 13th rib sites. Empty body weight has been determined by deducting the gut fill from the slaughter body weight, and the dressing percentage was calculated based on slaughter and empty body weight basis.To measure the rib-eye area of the carcass, the loin part was partitioned into fore and hind quarters between the 11th and the last ribs. The rib-eye area , CP (crude protein), and ash contents according to the methods of AOAC . NDF  was calculated as:TDMI (% BW) = (DMI (g)/BW) × 100 and/kg0.75) = DMI (g)/BW0.75TDMI (gDry matter intake (DMI) for each animal was calculated as.Where; CDMI = Commercial dry matter intake.DDMI = Duckweed dry matter intake.BW = Body weight.2.7.1The initial and final body weights of the rams were measured with the suspended weighing balance at the beginning and end of the experiment, respectively. Some of the measurements of body weight changes were calculated as:BWC = Final body weight (FBW) – Initial body weight (IBW)2.8μ is the overall mean,τi is the treatment effect, andεij is the random errorTo analyze the data, SAS version 9.0 , was use3The crude protein content of T3 24.87%) was higher than T2 (20.66%) and T1 (16.45%) . This wa.87% was 3.1The daily dry matter intake (DMI) was greater (P < 0.001) in T3 (914.375 g/day) than in T2 (870.5 g/day) and T1 (849.175 g/day) . This coThe total dry matter intake as a proportion of percent body weight and per unit metabolic body weight basis have no variation (P > 0.05) across the three treatments. The current TDMI as a proportion of percent body weight ranged from 3.74 – 3.83%, which was slightly higher than the value of 2.48–2.55% BW for Begait lambs kept under different feeding options reported by Kahsu, . The curThe dry matter intake per unit metabolic body weight (87.50–88.15 g/kg) obtained in the current finding agrees with the finding of Jalel  who recoThe daily CP intake was higher (P < 0.001) for T3 (144.77 g/day) than for T1 (114.10 g/day) and T2 (128.60 g/day). Such variations in CP intake could be attributed to the differences in the CP content of the supplemented duckweed to the commercial feed. The current finding is in line with the finding of Zetina et al.  who repoThe total NDF intake in T3 (473.42 g/day) was significantly higher (P < 0.001) than in T1 (409.07 g/day) and T2 (434.70 g/day), and the total OM and ADF intakes in T1, T2, and T3 have no significant variation (P > 0.05) for all treatments. The higher NDF in T3 may be due to its higher total dry matter intake. For both DMI and CP, the intake is higher in T3 and T2 than in the control group, and there is no refusal of experimental feeds. The study done by Darmy et al.  also fou3.2The final body weight, body weight change, daily gain, and feed conversion efficiency were higher (P < 0.001) for T3 than for T2 and T1 . This isThe feed conversion efficiency of the current study (0.05–0.08) was comparable with (0.05–0.04) and slightly higher than (0.03–0.09) reported by Gebrekidan , and Hay3.32) than T1 and T2 (Sheep fed on an increased level of duckweed (50%) supplemented diet are greater (P < 0.001) in slaughter body weight (24.43 kg), empty body weight 20.40 kg), hot carcass weight (10.27 kg), dressing percentage (DP) and rib-eye muscle area  between the supplemented and control groups. The dressing percentage based on empty body weight is 48.14%, 48.38%, and 50.31% in T1, T2, and T3, respectively. The current finding is in line with the finding of Ewnetu , for Men2, 7.11 cm,2 and 9.11 cm2 for T1, T2, and T3, respectively. As to Mathios et al. [The rib-eye muscle area is 6.08 cms et al. , and Wons et al. , supplem3.4In the current finding, duckweed supplementation did not distress the weight of blood, heart, and tongue (P > 0.05) . The oth3.5The weight of total inedible offals (TIO) are not influenced (P > 0.05) with duckweed supplementation . In the 4The study conducted to evaluate growth performance, carcass and non-carcass traits of Horro ram fed on commercial feed supplemented with duckweed showed that, with duckweed-supplemented commercial feed up to 50% level, it is possible to increase growth performance, quality, and yield of carcass traits of Horro ram, and thus could increase rate of return. Supplementary duckweed increases protein content of the ration with low cost to achieve improved feed intake, fast growth rate and body weight gain, and thus facilitate fattening practice of Horro ram. This indicates the possibility of using duckweed as an alternative protein feed resource to bridge the gap of feed scarcity and quality, especially in Ethiopia. Thus, for the further recommendation of this feed, testing for the presence of anti-nutritional factors has also an additional importance.Ashenafi Hunduma: Conceived and designed the experiment; Performed the experiment; Analyzed and interpretated the data and Wrote the paper.Debela Bayu: Conceived and designed the experiment; Analyzed and interpretated the data and Wrote the paper.Chala Merera: Conceived and designed the experiment; Contributed reagents, materials, analysis tools or data and Wrote the paper.Ulfina Galmessa: Conceived and designed the experiment, Analyzed and interpretated the data and Wrote the paper.Hirpessa Kabeta: Analyzed and interpretated the data and Wrote the paper.Data included in article/supp. Material/referenced in article.The author declares no conflict of interest.No additional information is available for this paper.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Escherichia coli (E. coli) O157:H7 and non-O157:H7 Shiga-toxin producing E. coli (STEC) serogroups. Sanitizer tolerance in STEC could be a public health concern as mitigation strategies against the pathogen might be compromised.The occurrence of antibiotic resistant (ABR) bacteria in foods is a growing public health challenge. We evaluated sanitizer cross-tolerance among ABR E. coli serogroups: O157:H7 , O121:H19 and O26:H11. Resistance to antibiotics was evolved chromosomally through incremental exposure to ampicillin (amp C) and streptomycin (strep C). Transformation using a plasmid was performed to confer resistance to ampicillin to generate amp P strep C.Resistance to ampicillin and streptomycin were evolved in P ≤ 0.05).The minimum inhibitory concentration (MIC) of lactic acid for all strains evaluated was 0.375% v/v. Analysis of bacterial growth parameters in tryptic soy broth amended with 0.0625% v/v, 0.125% v/v, and 0.25% v/v (subMIC) lactic acid indicated that growth correlated positively with the lag phase duration, and negatively with both the maximum growth rate and change in population density for all strains evaluated except for the highly tolerant variant- O157:H7 amp P strep C. Strains O121 NR (non-ABR), O121 amp C, O121 amp P strep C, O157:H7 H1730 amp C and O157:H7 H1730 amp P strep C were not inactivated after exposure to 1% and 2.5% v/v lactic acid for 300 s. No recovery of cells was observed after the strains were exposed to 5% v/v lactic acid for 300 s. ABR strains O157:H7 H1730 amp C and O157: H7 H1730 amp P strep C demonstrated a high tolerance to lactic acid (E. coli O157: H7 H1730 may improve tolerance to lactic acid. Increased tolerance may be discerned by evaluating growth parameters of bacteria in presence of sub-MIC levels of lactic acid.ABR in isolate  E. coli O157:H7 is the most commonly implicated STEC serogroup and is responsible for 40.3% of domestically acquired illnesses in the United States (US) (The contamination of foods by Shiga-toxin producing tes (US) . Emergintes (US)  and havetes (US) .Illnesses resulting from STEC infection could range from a mild, self-limiting gastroenteritis to the development of hemorrhagic colitis (bloody diarrhea) in 90% of patients and hemolytic uremic syndrome (HUS) in 5–15% of patients . HUS is E. coli O157:H7 in beef despite consistent use of sanitizers could be occurring because of the pathogens ability to adapt to acid stress through acid resistance systems (AR) (E. coli O157:H7 H1730 to lactic acid was linked to increased efflux pump activity in ABR E. coli O157:H7 (E. coli O157:H7 and STEC serogroups would result in a similar increase in tolerance to lactic acid.The continued foodborne outbreaks associated with ems (AR)  and efflems (AR) . Previou O157:H7 . Further O157:H7 ,b. It isE. coli O157:H7, E. coli O26:H11, E. coli O121:H19 would result in cross-tolerance to commercially used concentrations of lactic acid. Growth rate parameters of non-resistant parent strains and ABR variants were evaluated as predictors of elevated antibiotic tolerance.The objectives of this study were to evaluate if antibiotic resistance in outbreak associated isolates of E. coli were used in this study. E. coli O157:H7 H1730 was a human isolate from a lettuce outbreak and E. coli O157:H7 (ATCC 43895) was from a 1982 ground beef outbreak. Both isolates were obtained from the culture collection at the Center for Food Safety, University of Georgia. E. coli O121:H19 (strain TW08980) and E. coli O26:H11 (strain 3012-03) were from a 2016 Missouri flour outbreak and obtained from the Michigan State University STEC Center. Isolates were revived from frozen storage by transferring to Tryptic Soy Broth  and incubating at 37°C for 24 h. The bacterial strains were evaluated for antibiotic resistance to ampicillin and streptomycin by streaking on Tryptic Soy Agar  amended with 100 μg/ml of ampicillin, 100 μg/ml of streptomycin, and a combination of 100 μg/ml streptomycin and ampicillin. Plates were placed at an incubation temperature of 37°C for 24 h and observed for colony formation. Cultures that did not yield colonies in the antibiotic amended plates were considered to be susceptible to the antibiotics  of 0.8 and then placed in ice for 15 min. The cultures were then centrifuged at 1400 × g for 10 min to pellet the cells, and the supernatant discarded. The pelleted cells were washed three times with 15% ice-cold glycerol and stored at −20°C until use. The cells were transformed using the host range plasmid pGFPuv (Transformation of cells to take up a green fluorescence ampicillin resistance plasmid (GFP amp plasmid) was performed following the method described by d pGFPuv . Electrod pGFPuv .Stock cultures of the bacterial strains were prepared by streaking each strain to TSA or TSA + 100 μg/ml of antibiotics  and incubating at 37°C for 24 h. Colonies from overnight cultures were scraped from the plates with a sterile loop and suspended in phosphate-buffered saline . The bacterial population was adjusted to 6.42 ± 0.11 log CFU/ml.® 96 Well Flat Bottom, Corning LifeSciences Inc. ME, USA) were incubated for 24 h at 37 °C, and the growth kinetics were observed using the Bio-Tek Cytation 3 multi-mode plate reader . Conditions in the Bio-Tek Cytation 3 multi-mode plate reader were set as follows: the total runtime was set at 24 h with read intervals of 30 min, the shaker was set to an orbital shake every 10 s at a frequency of 283 cpm (3 mm), the read speed was set to Normal with a delay of 100 ms and the optical density was read at an absorbance of 600 nm. Un-inoculated blanks of TSB were used as a control for this experiment  for the antibiotic-resistant and non-resistant bacterial strains was determined using a 96 well plate broth dilution method described by periment .g using a Corning high speed microcentrifuge . The supernatant was discarded, and pellets were resuspended in 1 ml sterile deionized water (SDW). From the resuspended solution, 100 μl was transferred to 100 μl of 2 × TSB in 96-well plates  and incubated for 24 h at 37 °C. The plates were observed for turbidity to determine survival and regrowth after incubation by determining the ODnm600  using the BioTek Cytation multi-mode plate reader  was obtained from Xena Inc  and lactic acid solutions were prepared in TSB at concentrations of 0.5, 1, 1.5, 2, 2.5, and 5% v/v. The pH of lactic acid was determined using a pH meter  with a sensitivity of 0.01 and two-point calibration. The concentration at which lactic acid could prevent survival and regrowth of the STEC strains was considered as the bactericidal concentration. The bactericidal concentration of lactic acid for all the bacterial strains was evaluated as follows: Each 900 μl of 0.5, 1, 1.5, 2, 2.5, and 5% v/v lactic acid was inoculated with 100 μl of the bacteria for 300 s. Solution was centrifuged immediately after exposure for 1 min at 13000 × e reader .The growth rates for the different bacterial strains in TSB and the subminimum concentration of lactic acid were evaluated using the turbidimetric technique. The experiment was conducted in a 96 well microplate by inoculating 20 μl of 6.76 ± 0.71 log CFU/ml of bacteria to 180 μl of media. Growth rates were observed for 24 h at 37 °C using the Bio-Tek Cytation 3 image reader . Conditions in the Bio-Tek Cytation 3 image reader were set as described previously. Three biological and three technical replicates were performed, and strains exposed to TSB without the antimicrobial stress were used as controls.N is the bacterial population at a given time, N0 is the initial bacterial population, O.Dmin is the lowest O.D. value above the detection threshold, A is the logarithmic increase of bacterial population, L is the lag time, μ is the maximum growth rate, and t is time:The modified Gompertz model  modifiedA), lag phase duration in hours (L), and maximum growth rate in log CFU/h (μ max).The growth parameters assessed were change in bacterial population in log CFU/ml . The supernatant was discarded, and pellets were resuspended in 1 ml sterile deionized water (SDW). From the resuspended solution, 100 μl was transferred to 100 μl of 2 × TSB in 96-well plates  and incubated for 24 h at 37°C. The plates were observed for turbidity to determine survival and regrowth after incubation by determining the OD600 nm using the BioTek Cytation 3 multi-mode plate reader.A commercial food grade lactic acid (88% v/v) was obtained from Xena Inc  and lactic acid solutions were prepared in TSB at concentrations of 0.5, 1, 1.5, 2, 2.5, and 5% v/v. The pH of lactic acid was determined using a pH meter  with a sensitivity of 0.01 and 2-point calibration. The concentration at which lactic acid could prevent survival and regrowth of the STEC strains was considered as the bactericidal concentration. The bactericidal concentration of lactic acid for all the bacterial strains was evaluated as follows: Each 900 μl of 0.5, 1, 1.5, 2, 2.5, and 5% v/v lactic acid was inoculated with 100 μl of bacterial culture (6.89 ± 0.72 log CFU/ml) for 300 s. Solution was centrifuged immediately after exposure for 1 min at 13000 × g using the Corning LSE high speed microcentrifuge. The supernatant was discarded, and the pellets were resuspended in 1 ml 1 × PBS. Enumeration of bacteria was done using the droplet plate method where colony forming units (CFU) of cells within the perimeter of drop were used for enumerating bacterial populations from serially diluted samples. The number of colonies formed were counted after incubation for 24 h at 37°C. The limit of detection for the assay was 10 cells or 1.00 log CFU/ml.Overnight cultures of the bacterial strains grown either on TSA or TSA + 100 μg/ml of ampicillin, and TSA + 100 μg/ml of streptomycin and ampicillin were used to prepare 8 log CFU/ml of bacterial inoculum in 1 × PBS. From the suspended culture, 100 μl was transferred to 900 μl of TSB containing 2.5 or 5% lactic acid for 30 and 300 s. The solution was centrifuged immediately after exposure for 1 min at 13000 × All experiments were conducted in three biological and three technical replicates. Differences between the bactericidal concentration of lactic acid and decline in population of all bacterial strains evaluated after acid exposure were compared using the one-way analysis of variance (ANOVA). Significant differences between the means were compared using the Tukey’s Honestly Significant Difference (HSD) test at a 0.05 significance level, using JMP statistical software .n = 12) was inhibited by an MIC 0.375% v/v lactic acid.Growth of all STEC strains and their ABR variants (P ≤ 0.05)  .P > 0.05). Growth parameters of O157:H7 STEC (P ≤ 0.05). Generally, lactic acid concentration of 0.25% v/v significantly increased the average lag phase duration (L) by 1.15 h, decreased the average maximum growth rate (μmax) by 0.08 log CFU/h, and decreased the average change in bacterial population (A) by 0.3 log CFU/ml (P ≤ 0.05).Differences in antibiotic resistance (ABR) profile did not affect the MIC of lactic acid which was determined to be 0.375% v/v for all bacterial strains evaluated (:H7 STEC  and O121:H7 STEC  at 0.062P > 0.05) except at the μmax for O157:H7 43895 amp C in 0.0625% v/v lactic acid which was significantly different from the other variants (P ≤ 0.05) (P > 0.05).The L, μmax and A for bacterial strain O157:H7 43895 was not significantly different for both the parent strain and the ABR variants across all 3 sublethal lactic acid concentration ( ≤ 0.05) . The μmaP ≤ 0.05) and the highest A of 0.66 ± 0.01 log CFU/ml was observed for O121 amp P strep C (P ≤ 0.05) (P ≤ 0.05)  . In the  ≤ 0.05) .P < 0.05) (P ≤ 0.05). The A of 0.65 ± 0.01 observed for O26 NR in the control was significantly different from the A of 0.58 ± 0.01 observed for O26 amp P strep C (P ≤ 0.05).The difference between variants of strain O26:H11 was observed at 0.25% v/v lactic acid. The μmax of 0.14 ± 0.01 log CFU/h for O26 amp P strep C observed at 0.25% v/v lactic acid was significantly different from the μmax of 0.19 ± 0.01 log CFU/h observed for O26 NR ( < 0.05) . In the P ≤ 0.05) (P ≤ 0.05). The μmax of 0.21 ± 0.01 log CFU/h for O157:H7 H1730 amp P strep C was the smallest at this concentration (P ≤ 0.05) but O157:H1730 NR had the smallest A of 0.83 ± 0.04 log CFU/ml compared to the other O157:H7 H1730 strain variants (P ≤ 0.05). O157:H7 H1730 amp P strep C had the longest L of 5.58 ± 0.17 h at 0.125% v/v lactic acid and the smallest μmax of 0.23 ± 0.01 log CFU/h at 0.25% v/v lactic acid compared to the other O157:H7 H1730 strain variants (P ≤ 0.05).Bacterial strain O157:H7 H1730 amp P strep C had the longest L of 5.97 ± 0.04 h, 5.45 ± 0.08 h, 5.58 ± 0.17 h and 5.74 ± 0.03 h in the absence of lactic acid, 0.0625% v/v lactic acid, 0.125% lactic acid and 0.25% lactic acid, respectively, compared to all other strain variants evaluated ( ≤ 0.05) . At 0.06r = −0.18) and positive correlation with μmax (r = 0.21) (Increase in the lactic acid concentration of the medium correlated positively with the (L) and negatively with the (μmax) and (A) for all bacterial strains except O157:H7 H1730 amp P strep C that showed a negative correlation with L ( = 0.21) .P ≤ 0.05) (P ≤ 0.05). The most tolerant strains at 2.5% v/v lactic acid for 300 s were O157:H7 H1730 amp C 0.63 ± 0.17 log CFU/ml and O157 H7 H1730 amp P strep C 0.55 ± 0.29 log CFU/ml. The highest tolerance in 5% v/v lactic acid after 30 s exposure was observed in O157:H7 H1730 amp P strep C and O157:H7 H1730 amp C with 2.66 ± 0.14 and 2.56 ± 0.21 log CFU/ml decline in bacterial population, respectively, (P ≤ 0.05). All bacterial strains except O157:H7 amp C declined in population to below the limit of detection after exposure to 5% v/v lactic acid for 300 s  and exposure time (30 and 300 s) ( ≤ 0.05) . The hig ≤ 0.05) . After e ≤ 0.05)  and 4.92 ≤ 0.05)  respectior 300 s .E. coli O157:H1730 where strains amp C and amp P Strep C had higher tolerance to lactic acid than the parent strain and other combinations of resistance to ampicillin, streptomycin and the combination of ampicillin and streptomycin using both chromosomal adaptation and plasmid-based transformation  resulted in increased sensitivity to lactic acid (E. coli and requires further exploration. In this study, all three variants of E. coli O121 showed comparable levels of lactic acid tolerance by bactericidal concentration to O157:H7 H1730 amp C and O157:H7 H1730 amp P strep C.Tolerance to lactic acid due to ABR may be attributed to the activity of efflux pumps. In a previous study we observed that tic acid . Efflux tic acid . Efflux tic acid . ExpressCells that develop ABR were reported to spend an extended time in the lag phase . ResultsPrevious studies have reported variations in acid resistance between STEC serogroups. Among the serogroups evaluated in this study, previous studies have reported O121 to be the most acid resistant . In thisThe highest tolerance to lactic acid was observed in strains O121 NR, O121 amp C, O121 amp P strep C, O157:H7 H1730 amp C and O157:H7 H1730 amp P strep C confirming the high acid tolerance of O121 but also indicating that ABR in some STEC strains could improve acid tolerance.The results highlight the impact of antibiotic resistance on growth parameters in STEC and the risk of antibiotic associated tolerance to sanitizers.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.IO performed the experiments and drafted the manuscript. AM conducted analysis of growth parameters and edited the manuscript. GD conceptualized the study, performed the experiments, and drafted and edited the manuscript. All authors contributed to the article and approved the submitted version.


The present study was undertaken to investigate the effects of (m)VD-HPα on the sleep–wake cycle and power spectrum of cortical EEG in freely moving rats and the potential neurons in the brain activated by (m)VD-HPα. The results showed that 20.1 nmol of (m)VD-HPα i.c.v. administration increased non-rapid eye movement (NREM) sleep in the first 2 h section accompanied by an increase in EEG delta (0.5–4 Hz) activity. The (m)VD-HPα-induced NREM sleep enhancement was due to extended episode duration instead of the episode number. In addition, the effect of (m)VD-HPα (20.1 nmol) on sleep–wake states was significantly attenuated by an antagonist of the CB1 receptor, AM251  but not by the CB2 receptor antagonist, AM630 . In comparison with vehicle, (m)VD-HPα increased Fos-immunoreactive (-ir) neurons in the ventrolateral preoptic nucleus (VLPO), but reduced Fos-ir neurons in the lateral hypothalamus (LH), tuberomammillary nucleus (TMN), and locus coeruleus (LC). These findings suggest that (m)VD-HPα promotes NREM sleep via the CB1 cannabinoid receptor to probably activate VLPO GABAergic neurons, but inactivates the LH orexinergic, LC noradrenergic, and TMN histaminergic neurons.Hemopressin and related peptides have shown to function as the endogenous ligands or the regulator of cannabinoid receptors. The previous studies demonstrated that the endocannabinoid system played important roles in modulating several physiological functions such as sleep, olfaction, emotion, learning and memory, and reward behaviors. Mouse VD-hemopressin (α) [(m)VD-HPα], an 11-residue peptide derived from the α1 chain of hemoglobin, was recently presumed as a selective agonist of the CB Attempts were also made to identify the potential neuronal targets of (m)VD-HPα in the ventrolateral preoptic nucleus (VLPO), lateral hypothalamus (LH), tuberomammillary nucleus (TMN), and locus coeruleus (LC) involved in the sleep–wake regulation by analyzing c-Fos expression using ad libitum. All animals were cared for, and experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011 revision). The experimental protocol was approved by the Ethics Committee of Lanzhou University (permit number: SCXK Gan 2018-0002). All possible efforts were made to reduce the number of rats used and discomfort to the rats.Adult male Sprague–Dawley rats (8–10 weeks old), weighing 250–300 g, were purchased from the Experimental Animal Center of Lanzhou University . Upon arrival at the animal housing facility, they were kept in an automatically controlled room on a 12:12-h light/dark cycle  at an ambient temperature (22°C ± 1°C) and 50% relative humidity with food and water available Under pentobarbital sodium anesthesia , four stainless steel screw cortical electrodes (1 mm diameter) were screwed through the skull into frontal  and parietal  cortices to record electroencephalogram (EEG). Three silver wires were inserted into the dorsal cervical neck muscles to record electromyogram (EMG). The free ends of the electrode leads and silver wires were soldered into a seven-pin miniature plug. For i.c.v. injection, a guide cannula (0.6 mm diameter and 20 mm long) was stereotaxically implanted into the right lateral ventricle  of the rat according to the atlases of (m)VD-HPα (VDPVNFKLLSH) was synthesized by Shanghai Mocell Biotech Co., Ltd., Shanghai, PR China. Fresh (m)VD-HPα , Win55,212-2 , AM251 , and AM630  were dissolved in a 5-μL vehicle containing 5% cremophor, 5% dimethyl sulfoxide, and 90% saline before use according to After the experiments, 5 μL of methylene blue dye was injected into the ventricle via the guide cannula 5 min before the rats were decapitated under deep anesthesia with chloral hydrate. The brains were removed and frozen. Gross dissection of the brain was used to verify the site of drugs and vehicle administration; two rats whose microinjection sites were not located within the i.c.v. were excluded. Only the data from animals with dye dispersion throughout the ventricle were used.After surgery, the rats were housed singly in transparent barrels  and monitored using an infrared video camera in the recording chamber during both light and dark phases, and they were allowed to recover for 1 week. After the rats were connected to the recording cable attached to a slip ring for a habituation period of 2 days, a 24-h sleep–wake recording (20:00–20:00 h) was performed.EEG and EMG activities were amplified (2000×), filtered , and digitalized at a resolution of 256 and 128 Hz, respectively, using CED 1401 MK II . The behaviors of the rats during the light and dark phases in the chamber were monitored and recorded using an infrared video camera. Using a Spike 2 (CED) sleepscore script and with the assistance of spectral analysis by the fast Fourier transform (FFT), we visually scored polygraphic records by 30-s epochs for wakefulness (W), REM, and NREM sleep according to our previously described criteria validated for rats .n = 5) or vehicle (n = 5) i.c.v. administration, the rats were deeply anesthetized with pentobarbital sodium and perfused via the ascending aorta with 200 mL saline followed by 300 mL ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer . The brains were removed, post-fixed in 4% PFA overnight, immersed in 30% sucrose solution in 0.1 M PB at 4°C for 48 h, and then coronally sectioned (30 μm) on a cryostat  at −20°C.After 90 min of (m)VD-HPα  for 1 h. Then, the sections were incubated with a rabbit polyclonal antibody against c-Fos  in PBS containing 1% bovine serum for 72 h at 4°C on an agitator. After rinsing in PBS, the sections were incubated with a biotinylated goat anti-rabbit IgG  for 48 h at 4°C and then with horseradish peroxidase-conjugated streptavidin  for 24 h at 4°C. Following rinsing, the sections were immersed in 0.05 M Tris-HCl buffer (pH 7.6), containing 0.05% 3,3’ diaminobenzidine (DAB), 0.01% H2O2, and 0.6% nickel ammonium sulfate for 2–5 min at room temperature. The sections were mounted on gelatin-coated glass slides, dried, dehydrated, and covered with a coverslip, using DPX, for light microscopy.The floating sections were rinsed in 0.01 M PB saline , processed for 30 min in 0.3% HSections containing the VLPO , LH , TMN , and LC  were determined by the characteristics of their cytoarchitecture and peripheral white matter according to the rat atlases of post hoc Fisher’s least significant difference (LSD) test for sleep parameters and unpaired Student’s t-test for the number of Fos-ir neurons. The significance was set at p < 0.05.All data were expressed as means ± SEM. Statistical significance was analyzed by two-way repeated-measures analysis of variance (ANOVA) or one-way ANOVA followed by a Typical examples of the EEG power spectra, EEG, EMG, and the corresponding hypnograms from rats, respectively, given vehicle, 6.7, 13.4, and 20.1 nmol of (m)VD-HPα were presented in p < 0.01, p < 0.01, and p < 0.05) and with a concomitant decrease in W . Compared with vehicle, 13.4 nmol of (m)VD-HPα resulted in an increase in NREM sleep by 11.1% (p = 0.114), 9.8% (p = 0.291), 18.4% (p = 0.061), and 26.7% (p < 0.01) during 20:00–24:00 h (p < 0.05), meanwhile, decreased W by 13.6% in the 12-h dark phase (p < 0.05). The rat injection with 13.4 nmol of (m)VD-HPα had a 12.8% increase in NREM sleep (p = 0.226) and a 6.5% decrease in wakefulness during the 12-h dark phase (p = 0.319). Compared with vehicle, Win55,212-2 (2.5 nmol i.c.v.), a classic agonist of the CB1 receptor used as positive control, induced an increase in NREM sleep mainly during 20:00–21:00 h and 01:00–02:00 h of clock time and increased the amount of NREM sleep by 24.3% in the 12-h dark phase, which were similar to that induced by 20.1 nmol of (m)VD-HPα VD-HPα showed an increase in NREM sleep mainly during 20:00–22:00 h and 01:00–02:00 h of clock time in the dark period VD-HPα .p < 0.01) but not in REM sleep latency. In p < 0.05), 45.3% (p < 0.001), and 33.0% (p < 0.05), respectively, as compared to that in vehicle rats during the first 2 h post dosing; concomitantly, wakefulness decreased by 11.0% (p = 0.079), 26.4% (p < 0.01), and 20.2% (p < 0.05). Compared to (m)VD-HPα 13.4 rats, (m)VD-HPα 20.1 rats showed an increase in NREM sleep (p < 0.05) and a decreased wakefulness  or CB2 receptor antagonist AM630  was injected with 20.1 nmol of (m)VD-HPα. As typical examples of the EEG power spectra, EEG, EMG, and the corresponding hypnograms are shown in To identify whether the cannabinoid receptor antagonist blocked the effect of (m)VD-HPα, CBp < 0.05); however, AM630 did not alter these changes (p < 0.05) but not by AM630 VD-HPα-induced increase in NREM sleep and decrease in W during 20:00–22:00 h in the dark period ( changes . A cumulby AM630 .p < 0.05). In p < 0.01) through reducing episode duration (p < 0.01). Concomitantly, the decrease in W caused by (m)VD-HPα was blocked by AM251 (p < 0.01) mainly through increasing episode duration, although not reaching statistical significance (p = 0.141). However, AM630 did not antagonize the effect of (m)VD-HPα on NREM sleep latency, episode number, and duration of each stage (As shown in ch stage .p < 0.001) and decreased the number of Fos-ir neurons in the LH (p < 0.05), TMN (p < 0.05), and LC .Compared to vehicle, the injection of (m)VD-HPα increased the number of Fos-immunoreactive (-ir) neurons in the VLPO VD-HPα decreased the NREM sleep latency and promoted NREM sleep through prolonging the mean episode duration. The effects were accompanied by an enhancement of cortical slow activities and a suppression of fast rhythms –3. Thesest AM630 –6.1 receptor antagonist rimonabant commonly reported insomnia and poor sleep quality VD-HPα was primarily due to increased NREM bout length , and thest AM251 . The prost AM251 , and sevt length . In addiEM sleep  to furthEM sleep , and lotEM sleep . Interes effects ; thus, w1 receptor is expressed in many brain regions, especially, its distribution in the pons and hypothalamus is of fundamental importance here VD-HPα directly activates the GABAergic neurons in the VLPO or directly inhibits the orexinergic, noradrenergic, and histaminergic neurons in LH, LC, and TMN, respectively, in the present study. Nevertheless, our findings suggest that (m)VD-HPα, the peptidic endocannabinoid, promotes NREM sleep via the CB1 cannabinoid receptor to probably inactivate the neurons in LH, LC, and TMN and activate the neurons in the VLPO.To identify the target neurons through which (m)VD-Hpα promotes NREM sleep, we labeled activated neurons by staining c-Fos, one product of the immediate early gene that is expressed in association with neuronal activation . The resnce here , given tsynapses , where tsynapses . These o1 receptor antagonist AM251 but not by CB2 receptor antagonist AM630. (m)VD-HPα markedly increases the number of Fos-ir neurons in the VLPO and decreases that in the LC, LH, and TMN. These results indicate that (m)VD-HPα promotes and stabilizes NREM sleep via the CB1 cannabinoid receptor to probably activate VLPO GABAergic neurons and inactivate the LH orexinergic, LC noradrenergic, and TMN histaminergic neurons.Central administration of peptidic endocannabinoid (m)VD-HPα significantly enhances NREM sleep and reduces wakefulness. (m)VD-HPα-induced NREM sleep enhancement was due to the extended episode duration instead of the episode number. These effects are significantly blocked by CB


Baked goods manufacturing parameters and fermentation conditions interfere with the nutrients content and affect their gastrointestinal fate. Pinsa Romana is a type of pizza that, recently, has been commercially rediscovered and that needed elucidation from a nutritional and digestibility perspective. In this study, six types of Pinsa Romana (five made with indirect method and one produced with straight dough technology) were characterized for their biochemical and nutritional features. Several variables like indirect (biga) Pinsa Romana production process, fermentation time and use of sourdough were investigated. The Pinsa Romana made with biga including sourdough and fermented for 48 h at 16 °C ((PR_48(SD)) resulted in the lowest predicted glycemic index, in the highest content of total peptides, total and individual free amino acids and gamma-amino butyric acid (GABA), and in the best protein quality indexes . The static in vitro digestion showed that the digesta from PR_48(SD) confirmed a reduced in vitro glycemic response after intake, and it showed a lower bioavailability of hydrophilic peptides. Furthermore, the inclusion of sourdough in biga enhanced the bioavailability of protein-related end-products including human health promoting compounds such as essential amino acids. Baked goods refer to a wide range of food with different textures and flavors that can be designed to provide a variety of nutritional properties ,2. In paThe production process of PR requires a long leavening, commonly from 24 to 72 h . Baker’sIn this context, food absorption, protein and starch digestibility depend on mechanical, chemical, and biochemical events that allow the disruption of nutrients and bioactive compounds . As a reFirst, our study aimed at assessing how different PR-making processes (direct or indirect) affect nutritional properties and digestibility. Additionally, the combined use of biga and sourdough, and the length of the fermentation stage in the yeasted biga were assessed to highlight the conditions that affect the digestibility and the overall quality of the PR. To that end, the static INFOGEST in vitro digestion method  and stat(Triticum aestivum L.) flour , stone ground wheat (Triticum aestivum L.) flour , rice (Oryza sativa L.) flour  and soy (Glycine max L. (Merr.)) flour  were used. The gross chemical composition of the flours used for making sourdoughs, biga and PRs were as follows: soft wheat flour, protein 12.1 g/100 g, fats 1.1 g/100 g, carbohydrates 67.1 g/100 g, fibers 4.0 g/100 g; stone ground wheat flour, protein 10.7 g/100 g, fats 1.5 g/100 g, carbohydrates 67.0 g/100 g, fibers 5.3 g/100 g; toasted full fat soy flour, protein 40.5 g/100 g, fat 20.8 g/100 g, carbohydrates 10.6 g/100 g, fibers 14.5 g/100 g; rice flour, protein 8.3 g/100 g, fats < 1.5 g/100 g, carbohydrates 76.0 g/100 g, fiber 1.0 g/100 g.Commercial soft wheat A spontaneous mother dough was prepared by mixing 50% of stone ground wheat flour and 50% of tap water and fermented for 18 h at 30 °C. The procedure was repeated five times until the mother dough was stable, and it was continuously subjected to a maintenance process . The final sourdough (SD) was prepared by mixing soft wheat flour, tap water and the stable mother dough at the ratio of 37.5%, 37.5% and 25%, respectively, and it was used for the production of biga and PRs. The SD was mature after fermenting for 4 h at 30 °C. Before use, SD was kept for 8 h at 4 °C.w/w) and the sourdough-biga was fermented for 48 h. The different biga were used to produce five different PRs as detailed in w/w), olive oil  and salt ) and addition of the baker’s yeast biga fermented for 24 or 48 or 72 h . One PR was produced with the same raw ingredients and the addition of both the baker’s yeast-biga fermented for 48 h and 19% (w/w) of SD (PR_48+SD). The last PR was manufactured with the same raw ingredients and the addition of the sourdough-biga (PR_48(SD)). A control thesis was also produced with the same raw materials but without the use of either biga or SD (PR_RT). Five types of biga that differ in the time of fermentation  and/or addition of SD were prepared. The temperature of fermentation was 16 °C for all types of biga. When SD was included in the recipe, it was added at 5% (w/v) and homogenized with a Classic Blender  to obtain a mother suspension. Decimal serial dilutions were prepared in 9 mL of sterile physiological solution. LAB were enumerated through the plate count method after the incubation at 30 °C for 48 h using modified MRS agar  medium, prepared by the addition of 0.5% (w/v) maltose and 0.5% (w/v) yeast extract to standard MRS medium and supplemented with cycloheximide (0.1 g/L) . Yeasts cell density was estimated on Sabouraud dextrose agar  medium supplemented with chloramphenicol (0.1 g/L)  after incubation at 30 °C for 48 h. A control dough produced without biga and SD was also considered for such characterizations. After growth, colonies from SD with different morphologies were isolated from the highest plate dilution and identified by the partial sequencing of the gene encoding for 16S rRNA (for LAB) and for 26S rRNA (for yeasts) as reported by [SD and bigas pH was determined through a pH meter  with a food penetration probe. To assess the cell density of lactic acid bacteria (LAB) and yeast, ten grams of each biga or SD were added to 90 mL of physiological solution , PR_48+SD, PR_72 and PR_RT) was carried out according to the consensus method developed within a large European framework (COST Action InfoGest) with some modifications ,24,25. Fw/w) with simulated salivary fluid  solution 2, 0.15 mM; (NH4)2CO3, 0.06 mM). The suspension was added with 0.75 mL of amylase solution (1500 U/mL of SSF) and CaCl2 0.3 M solution (41.98 μL). The final mixture was mixed for 2 min at 37 °C. Then a stomach phase followed in which the oral bolus was diluted with a mixture mimicking the gastric content. The stomach phase incubation started by adding 82.02 mL of gastric juice (0.04 M KCl and 0.24 M NaCl). The suspension was supplemented with 6.07 mL of 2% pepsin solution (≥400 U/mg) and 0.68 mL of lecithin solution (0.17 mM). The incubation lasted for 2 h at 37 °C, with a pH gradient from 5.5 to 2.0. Finally, a small intestinal phase took place in which the gastric chyme (140 mL) was added with 50.00 mL of simulated pancreatic juice ), 4.5 mL of trypsin solution (50 mg of trypsin 5000 USP-U/mg in 5 mL of 1 mM HCl), 5.6 mL of chymotrypsin (100 mg chymotrypsin ≥ 1000 USP-U/mg in 10 mL of 1 mM HCl), 180 mg of lipase  and 300 mg of hog α-amylase (50 U/mg), and 350 μL of 0.3 M CaCl2. The entire mixture (200 mL) was incubated for a further 3 h at 37 °C under static dialysis with a 14 kDa membrane in 400 mL of 0.04 M NaHCO3 dialysis solution (pH 7) within a bioreactor, according to [They include an initial oral phase in which PR samples were freeze-dried, ground, and diluted 1:2  and fats of different PRs were determined according to ISTISAN Report 96/34 . Total cProteins, lipids, carbohydrates, and ash were expressed as % of fresh weight (fw).The pH of different PRs was determined as mentioned in paragraph 2.4. Total titratable acidity (TTA) was also measured. Briefly, 10 g of the six PRs was homogenized with 90 mL of distilled water and TTA was expressed as the amount (mL) of 0.1 M sodium hydroxide (NaOH) required to neutralize the solution  according to the official AACC method 02–31.01 . Phytic The evaluation of IVPD was carried out on PR_24, PR_48, PR_48(SD), PR_48+SD, PR_72 and PR_RT according to the method of Akeson and Stahmann , with soThe digested protein fraction, deriving from 1 g of PR_24, PR_48, PR_48(SD), PR_48+SD, PR_72 and PR_RT samples, was added to 5.7 M HCl, under a nitrogen steam and incubated at 110 °C for 24 h. After freeze-drying, the hydrolysate was resuspended in lithium citrate buffer, pH 2.2, and filtered through a Millex-HA 0.22 µm pore size filter . The modified AOAC method (2005)  was usedThe Essential Amino Acid (EAA) index, which estimates the quality of the test protein, using its EAA content as a criterion , was calThe Biological Value (BV) indicates the utilizable fraction of the test protein , and wasThe Protein Efficiency Ratio (PER), which estimates the protein nutritional quality based on the amino acid profile after hydrolysis, was determined using the equation developed by Ihekoronye :(4)PER =The Nutritional Index (NI) normalizes the qualitative and quantitative variations of the test protein compared to its nutritional status. NI was calculated using Crisan and Sands equation  which coThe sequence of limiting EAA, corresponding to the list of EAA having the lowest Chemical Score (CS), which estimates the amount of protein required to provide the minimal EAA pattern for adults, was calculated using the equation of Block and Mitchell .The degree of starch digestion was expressed as a percentage of potentially available hydrolyzed starch according to . The pGIWater/salt-soluble extracts (WSE) from PR_24, PR_48, PR_48(SD), PR_48+SD, PR_72 and PR_RT were prepared according to . Lactic + series Automatic Amino Acid Analyzer , equipped with a Li-cation-exchange column , as described by [For FAA analysis the WSE of PRs and the liquid-phase of the corresponding digesta were used with a Biochrom 30ribed by .o-phthaldialdehyde (OPA) assay [v/v) trifluoroacetic acid (TFA) and centrifuged at 10,000× g for 10 min, and the supernatant was filtered through a 0.22 μm pore size filter and loaded into the column. The elution process was carried out by increasing the acetonitrile (CH3CN) concentration linearly from 5% to 46% between 16 and 62 min, and from 46% to 100% between 62 and 72 min at the flow rate of 1 mL/min using a mobile phase composed of water and CH3CN containing 0.05% TFA [The concentration of peptides was determined both in the WSE from the PRs , PR_48+SD, PR_72 and PR_RT) and in liquid-phase of the corresponding digesta , D-PR_48+SD, D-PR_72 and D-PR_RT) using A) assay . In addi.05% TFA .n = 3). Data were subjected to Principal Component Analysis (PCA), and to one-way ANOVA; a comparison of sample means was obtained by Tukey’s procedure with a 95% confidence interval (p < 0.05). The FAA overall dataset was used to generate a graphic output (pseudo-heatmap) to assess the clustering of samples from the single variable’s matrix. Statistical analysis was performed on Statistica 8.0  and XLSTAT® software .Data used for statistical elaboration were the means of three independent analyses ± standard deviations , while the same biga fermented for 24 h and 48 h resulted in 6.40 ± 0.21 and 6.35 ± 0.17 Log CFU/g, respectively. The cell density of LAB in the control dough produced without biga and sourdough to produce PR_RT was <5 Log CFU/g. SD  used for making PR_48(SD) and PR_48+SD, had a LAB and yeasts cell density of 8.06 ± 0.01 and 7.33 ± 0.13 Log CFU/g, respectively . Seven iThe nutritional labels of the six different PRs are reported in The macronutrient content showed no marked variations between the PRs analyzed. Specifically, total fat content ranged from 5.4 to 5.9 g/100 g, total carbohydrates ranged from 75.8 to 77.4 g/100 g and total protein content was between 14.8 and 16.0 g/100 g. Even slightly, PR_48(SD) showed the lowest total fat content (5.4 g/100 g) and the highest total carbohydrate concentration (77.4 g/100 g), while PR_48+SD showed the highest total fat content (5.9 g/100 g). Both PRs produced with SD showed the lowest total protein amount (14.8 g/100 g). The consumption of 100 g of PRs corresponded to an intake between 406 (PR_48) and 417 (PR_48+SD) Kcal.p < 0.05) among all samples  reached the lowest pH value  compared to PR_48(SD) was found when SD was included only in the final dough . PRs made with biga including only baker’s yeast  showed a pH value significantly (p < 0.05) higher compared to that of those produced with SD (PR_48(SD) and PR_48+SD). As expected, in these PRs, the longer fermentation time (PR_48 and PR_72) resulted in lower pH (p < 0.05) compared to PR_24. The highest (p < 0.05) values of pH were found for the PR made without biga and sourdough (PR_RT) together with PR_24 . Accordingly, TTA values showed an opposite trend (Lower acidification (te trend . Phytic te trend .p < 0.05) lower pGI (63.9 ± 0.1) (p > 0.05) differences were found in EAA and BV indexes among PRs, ranging from 68.97 ± 3.73 to 75.85 ± 5.70 and from 63.48 ± 4.11 to 70.98 ± 3.98, respectively. The inclusion of SD in biga led to a 58.1% increase (p < 0.05) in the PER index in PR_48(SD) compared to PR_RT. Accordingly, the NI was significantly higher (p < 0.05) in PR_48(SD) and PR_72 .The recipe and the processing conditions did not affect the percentage of IVPD of the leavened baked goods, while the PR prepared with the addition of SD in the biga (PR_48(SD)) was the only one with significant (9 ± 0.1) . The proThe inclusion of SD in PR_48(SD) and PR_48+SD favored lactic and acetic acid production .p < 0.05) concentration of lactic and acetic acids  followed by PR_48+SD, where the SD was included only in the final dough . The other PRs showed lactic acid concentrations ranging from 2.3 to 2.9 mM with no statistical (p > 0.05) differences. Contrarily, acetic acid was not found in PR_24 and PR_48, while PR_72 contained a low amount (1.2 ± 0.3 mM).PR_48(SD) showed the highest (p < 0.05) content of lactic acid compared to D-PR_48+SD , while in the digesta from PR_24, PR_48, PR_72 and PR_RT lactic acid was under the detection limit. Acetic acid was not found in the digesta from all PRs.The digesta from PRs produced with biga including SD (D-PR_48(SD)) showed a higher (p < 0.05) glucose concentration  compared to PR_48 and PR_24. On the contrary, PR_RT, made without biga or sourdough, showed the lowest (p < 0.05) content of glucose (p < 0.05) lower in PR_48(SD) (2.3 ± 0.1 mM) and PR_48+SD (4.5 ± 0.1 mM), and significantly (p < 0.05) higher in PR_72 (14.0 ± 0.1 mM) compared to other PRs. Mannitol was found only in the PRs produced with SD. However, in PR_48(SD) the concentration (41.8 ± 0.1 mM) was significantly (p < 0.05) higher compared to PR_48+SD (6.2 ± 0.1 mM). Furthermore, maltose was present in all PRs, with the highest (p < 0.05) values in PR_RT (74.7 ± 0.1 mM), followed by PR_72 (61.0 ± 0.1 mM) and PR_48(SD) (60.6 ± 0.1 mM) (The use of SD both in biga (PR_48(SD)) and in the final dough (PR_48+SD), and the longer fermentation time (PR_72) resulted in higher ( glucose . Fructos 0.1 mM) .p < 0.05) different compared to D-PR_48+SD . Moreover, the different manufacturing parameters of PRs did not interfere (p > 0.05) with the maltose content in the PRs digesta that ranged from 741.2 ± 15.2 to 665.8 ± 41.4 mM. The glucose content in D-PR_48(SD) was significantly higher (p < 0.05) compared to D-PR_RT and D-PR_48. The glucose level in digesta was 188.5 ± 3.3 and 126.9 ± 22.7 mM in D-PR_48(SD) and D-PR_RT, respectively.Carbohydrates were evaluated also in the digesta from PRs to assess their fate along the GI tract. After the in vitro small intestinal digestive (D) simulation, the level of simple carbohydrates strongly changed. The fructose concentration in D-PR_48, D-PR_48(SD) and D-PR_48+SD was under the limit of quantification (< LOQ). Accordingly, after in vitro small intestinal digestion the concentration of mannitol showed the same trend already observed in PR_48(SD) and PR_48+SD. Indeed, the amount of mannitol in D-PR_48(SD) was statistically (p > 0.05) differences among the six PRs, as well as for the digested PRs.Total dietary fiber content was assessed to evaluate the behavior of non-digestible compounds through in vitro digestion. The value of total dietary fibers before digestion ranged from 15.4 ± 1.7 to 13.7 ± 0.7% d.m., while after digestion it ranged from 16.6 ± 1.1 to 13.4 ± 0.9% d.m., with no statistical (p < 0.05) (p > 0.05) were found in the content of RS after the digestion of PRs.Resistant starch in no-digested PRs ranged from 0.7 ± 0.1 (PR_48+SD) to 1.1 ± 0.1 g/100 g d.w. (PR_24) and only PR_24 and PR_48+SD differ significantly ( < 0.05) . No statp < 0.05) abundance in PR_48(SD) (12.7 ± 0.2 g/Kg) compared to PR_48+SD (12.0 ± 0.1 g/Kg) and PR_72 (11.4 ± 0.1 g/Kg), while PR_24, PR_48 and PR_RT had the lowest (p < 0.05) content (p > 0.05) total peptide amount to D-PR_48+SD , while the value was higher (p < 0.05) than D-PR_72 (165.8 ± 1.3 g/Kg).Overall, the inclusion of SD in biga resulted in the highest level of protein hydrolysis. Specifically, total peptides had higher   at different elution steps, reported as the concentration of CHN > 41%) .3CN < 19%), the highest relative values were 22.6% for PR_72 and 19.1% for PR_48(SD), while the relative lowest value was 5.8% for PR_48+SD. Conversely, increasing the CH3CN gradient (19% < CH3CN < 41%) the relative highest release of peptides was found in PR_48+SD (94.2%). No RP-FPLC peptide profiles were detected when the gradient of CH3CN was higher than 41% for all PRs. The analysis showed the opposite behavior in the digesta. In particular, the AUC of D-PR_RT (3611.1 mAU·min) was relatively higher than those found for the other digested PRs, followed by the digesta of PRs manufactured with longer fermented biga produced with only baker’s yeast . The D-PR_48(SD) showed the lowest relative percentage of the total area in the hydrophilic zone, whereas it displayed the highest value (28.9%) in the elution interval from 19 to 41% of the CH3CN gradient. Differently from no-digested PRs, all digested PRs showed the presence of a peptides profile in the more hydrophobic zone (CH3CN > 41%).In no-digested PRs, the analysis showed a total area under the curve (AUC) from 2196.9 mAU·min to 3693.0 mAU·min (PR_RT and PR_48(SD), respectively). In the more hydrophilic zone (CHp < 0.05) higher (557.14 ± 14.72 mg/kg) than other PRs, followed by PR_72 (425.56 ± 11.29 mg/kg). In general, most EAA resulted higher in the PRs produced with SD or longer fermentation time (PR_48(SD), PR_48+SD and PR_72). In particular, the concentration of Val, Met, Ile, Leu, Phe, and Trp were statistically (p < 0.05) higher in PR_48(SD). Accordingly, the content of γ-aminobutyric acid (GABA) was the highest (p < 0.05) in PR_48(SD) (66.92 ± 0.97 mg/kg), followed by PR_72 (59.67 ± 0.87 mg/kg).All individual FAA profiles and the total content before (mg/Kg) and after (mg/L) in vitro small intestinal digestion are reported in p < 0.05) total FAA, followed by D-PR_72 (1068.14 ± 26.98 mg/L). Anyway, the total FAA amount in D-PR_48(SD) was ca. 19.4% higher than D-PR_72. Overall, EAA  on digesta were significantly (p < 0.05) higher in D-PR_48(SD), D-PR_48+SD and D-PR_72 compared to the other samples. On the contrary, D-PR_48, D-PR_24 and the digesta of the control sample (D-PR_RT) showed the lowest content for individual FAA.After digestion, D-PR_48(SD) (1275.30 ± 51.92 mg/L) and D-PR_48+SD (1205.88 ± 81.57 mg/L) showed the highest  were used to differentiate the six PR samples. Before digestion, PC1 and PC2 explained 51.04% and 27.94%, respectively, of the total variance A. PCA clBecause of the poor digestibility found in the consumption of baker’s yeast products ,45,46, PUnder our experimental design, the PR made with sourdough-biga fermentation showed the best nutritional profile among the six investigated samples, in agreement with literature dealing with different sourdough baked goods ,49,50. IF. sanfranciscensis) [Fructose in PR_48(SD) was almost totally converted to mannitol and, although milder, this effect was found also in PR_48+SD. This can be due to the heterofermentative LABs isolated in the SD  . The proscensis) . Furtherscensis) .As the IVPD analysis did not highlight differences among PRs produced, the EAA index, BV index, PER and NI nutritional indexes were deeply investigated to identify if the production conditions could enhance the protein quality. While BV and EAA indexes were similar among the six PRs, also considering the quantity variations in the protein portion , the ferThe inclusion of sourdough in biga (PR_48(SD)) allowed a significant increase in restrictive EAA, with the only exception of Lys and Thr which could be used as a growing substrate by fermenting microorganisms . The conAiming at investigating macronutrient fate along the GI tract and their changes to determine manufacturing parameters that enhance the digestibility of PRs produced with biga, static in vitro digestion was performed. The control sample, PR_RT, was digested under the same conditions.As the contribution of the gastric phase to the overall digestion of glucose-based polysaccharides is negligible ,65 and, The monitoring of simple sugars after dialysis was conducted to assess the glycemic response and to extend our findings on the starch digestibility of PRs. The generation of glucose in D-PR_48(SD) after 3 h of small intestinal dialysis was 48.5% higher than D-PR_RT. This finding is coherent with pGI data and agreed with previous studies on polysaccharides matrices which reported an analogous trend . TherefoThe protein digestibility was assessed by evaluating the soluble digesta nitrogen fraction from peptides and FAA. The OPA method highlighted the higher total peptide content in the digesta of the PR produced with sourdough (D-PR_48(SD)) compared to that of PRs manufactured with baker’s yeast biga. Since the total peptide content did not consider the hydrophobic variations that could be present in the digesta, the peptide release was studied also by using the RP-FPLC analysis. The digesta of PR_48(SD) showed reduced bioavailability of hydrophilic peptides (visible in the first elution step), which can probably be attributed to easier absorption at the small intestine level as a result of consumption of food that was pre-digested during the fermentation process .The inclusion of sourdough enhanced the levels of total FAAs at small intestinal levels, which are typically linked to improved overall health .The digesta analysis showed a positive effect of biga sourdough (D-PR_48(SD)), which increased the level of total FAAs compared to the reference (D-PR_RT). In contrast to what was found in the non-digested counterparts (PRs), after intestinal digestion in vitro, the addition of sourdough in the final dough (D-PR_48+SD) had a similar impact as the inclusion of sourdough in biga (D-PR_48(SD)) in increasing total FAA. Leu and Arg were the most abundant FAA at the end of PR_48(SD) digestion (D-PR_48(SD)), followed by Phe and Tyr .The use of a biga including sourdough for the manufacturing of PRs resulted in improved carbohydrate digestibility and protein quality after in vitro digestion. The combination of two bakery biotechnologies, SD and biga fermentation, had the capability to reduce the pGI and the glucose absorption and to improve the generation of the protein-related health-promoting compounds and the protein quality indexes. Although the digestibility of baked goods is also related to satiety and gastrointestinal symptoms after consumption , such da


Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold  for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 β2β3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure–activity relationships of α-conotoxins, which may help in the design of more selective tools.α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely  It is further divided into five archipelagos: the Society Islands, the Marquesas Islands, the Tuamotu Islands, the Gambier Islands, and the Austral Islands. Due to their geographical isolation, the islands of Polynesia have developed unique flora and fauna, with many endemic species found nowhere else in the world. Endemic cone snails of Polynesia are remarkable examples of this biodiversity, as these species are rare and unique to this region. These snails are characterized by their unique shells, which are often brightly colored and beautifully patterned. Among the most iconic are Conus gauguini (named after French painter Paul Gauguin) and Conus adamsonii, which are favorites among shell collectors. Polynesia is an archipelago made up of over 100 islands spread over an area of 4 million kmConus magus venom and approved by the FDA in 2004 for the treatment of chronic pain. It constitutes one of the very few drugs derived from marine natural compounds BuIA (IC50 = 58.1 nM) LvIC (IC50 = 19 nM) [50 value of 177.3 nM at the α6/α3β2β3 combination . In contrast, it shows potency that is at least 8-fold lower at α3β2 (IC50 = 1375 nM) and micromolar potency (>10 μM) at all other subtypes. This makes it the first 4/4 framework α-conotoxin selective for the α6β2 interface.LvIC inhibited only α6/α3β4 with a micromolar potency  as α-MI (IC50 = 12 nM) and α-GI (IC50 = 20 nM). However, while α-MI and α-GI showed no effects on α2β2, α3β2, α3β4, α4β2, α4β4 and α7 receptors [50 = 777.2 nM), α3β2 (IC50 = 988.9 nM), the closely related α6/α3β2β3 , α7 (IC50 = 5158 nM), α2β2 (IC50 = 6474 nM), and α3β4 (IC50 = 7912 nM) neuronal nAChRs. GaIA belongs to the α-3/5 subfamily and is closely related to α-MI and α-GI. These α-conotoxins have been widely studied for their high selectivity towards different interfaces of the muscle type and Torpedo nicotinic receptors. Likewise, the newly synthesized peptide GaIA shows nanomolar potency at the muscle-type α1β1γδ nAChR with a similar ICeceptors , GaIA reThus, AdIA and particularly GaIA display a wider pharmacological profile on nAChR subtypes compared to the other characterized members of this family and might provide a template for further structure–activity studies.Acm, acetamidomethyl; ACN, acetonitrile; DCM, dichloromethane; DMF, N,N-dimethylformamide; DIPC, disopropylcarbodiimide; DTT, dithiothreitol; DTC, diethyldithiocarbamate; DSF, disulfiram; eq, equivalent; ESI-MS, electrospray ionization-mass spectrometry; FA, formic acid; Fmoc, fluorenylmethoxycarbonyl; GSH, reduced glutathione; GSSH, oxidized glutathione; IPA, isopropanol; LC/MS, liquid chromatography/mass spectrometry; nAChRs, nicotinic acetylcholine receptors; NBzl, 2-nitrobenzyl; RP-HPLC, reversed-phase high-performance liquid chromatography; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid; TIS, triisopropylsilane; Tris, 2-amino-2-(hydroxymethyl)propane-1,3-diol; Trt, trityl; UV, ultraviolet. 2O/DTT (88/2/5/5) (v/v) under agitation for 2 to 3 h. The reaction mixture was filtered, and the filtrate was dropwise added to cold diethyl ether to give a crude peptide precipitate and centrifuged. This synthetic method was applied to all synthetic conotoxin precursors, which were obtained with correct yields.The linear precursors of conotoxins were synthesized on a Liberty Blue peptide synthesis instrument  at a scale of 0.1 mmol. The synthesis was conducted on a Rink-amide resin with a loading capacity of 0.34 mmol/g. The standard amino acid coupling cycle is as follows: 2.5 mL of amino acid (5 eq) are added to the reactor, activated with 1 mL of disopropylcarbodiimide (DIPC) (5 equiv.) and 0.5 mL of Pure Oxyma (10 equiv.). The resulting mixture was heated at 90 °C in the microwave for 2.15 min. After each coupling reaction, the resin was subjected to a deprotection cycle of 2 min at 90 °C under microwave with a solution of DMF/Piperidine (80/20) followed by washing with DMF. Side-chain (except Acm and Nbzl) deprotection and cleavage from the resin were carried out using 10 mL of TFA/Tis/H16 s cm−3 photons) in a photochemical 16 chamber reactor under ice-cooled conditions for 8–10 min. Purification was performed by using a semi-preparative C18 column.The lyophilized linear conotoxin peptide was dissolved (0.5 mM) in 6 M Gn·HCl buffer, pH 7, and treated with 10 equiv. DSF for 10 s at 37 °C. Subsequently, the reaction mixture pH was adjusted to 6 using 0.1 M HCl and exposed to UV radiation at 350 nm  followed by 10 equiv. of DSF, and the pH of the reaction was adjusted to 6.0 using 0.1 M HCl to complete the reaction.The lyophilized linear conotoxin peptide (AdIA or GaIA) was dissolved (0.5 mM) in 6 M Gn·HCl buffer, pH 7, and treated with 10 equiv. DSF for 10 s at 37 °C. Subsequently, the reaction mixture pH was adjusted to 1 using 0.1 M HCl, treated with 10 equiv. PdClThe lyophilized linear conotoxin peptide (0.25 mM) was dissolved in the selected buffer solution and subjected to agitation for 48 h. Upon completion of the reaction, the mixture was acidified to pH 3 using a 10% formic acid solution for subsequent analyses. Based on Gyanda et al. , the sam18, 3.5 μm, Column   at a flow rate of 1.2 mL/min, coupled with a diode array. All solvents used were HPLC-grade. Analytical HPLC was performed on a Dionex Ultimate 3000 , using analytical XBridge Peptide BEH CThe LC–MS system consists of a Thermo Fisher Scientific LC–MS device, an Accela HPLC coupled to a QFleet fitted with an electrospray ionization source, and an ion-trap analyzer. All analyses were performed using a Kinetex 2.6 μm C18 column (150 × 3.00 mm) from Phenomenex Inc.  in linear gradient mode from 2% to 60% over 30 min with a flow rate of 0.5 mL/min .The UHPLC/HRMS system consists of a Vanquish UHPLC  coupled to a QTOF Maxis II mass spectrometer , source electrospray ionization mode, ESI+. All analyses were performed using a bioZen™ 2.6 μm Peptide XB-C18 column   in linear gradient mode from 2% to 50% over 50 min with a flow rate of 0.3 mL/min  at 40 °C. Acetonitrile and formic acid RS for LC–MS ; ultra-pure water from PURELAB Chorus 1 .2O, 0.1% FA) into buffer B (acetonitrile + 0.1% FA). The column used was a GRACE Vydac C-18 column   at a flow rate of 3 mL/min.Semi-preparative purification of cyclic peptides was performed using a Waters 1525 chromatography system fitted with a Waters 2487 tunable absorbance detector with detection at 214 nm and 254 nm. Purification was performed by eluting buffer A  and kept at the core facility animal models of the biomedical center of the LMU Munich (Az:4.3.2-5682/LMU/BMC/CAM) in accordance with the EU Animal Welfare Act. Plasmids encoding the neuronal nAChR subunits  were provided by Jim Patrick  and subcloned in pNKS2. Rat muscle α1, β1, γ, and δ subunits in pSPOoD were provided by Veit Witzemann . The α6/α3 chimera was generated in pNKS according to ,36,37. R2, 1 mM MgCl2, 5 mM HEPES, and pH 7.4) containing 5 μg/mL gentamicin. After 1–4 days, two-electrode voltage clamp recordings were performed at −70 mV with a Turbo Tec 05X Amplifier  and CellWorks software (version 6.2.2). Electrode resistances were less than 1 MΩ, and currents were filtered at 200 Hz and digitized at 400 Hz. The recording solution ND96, with or without 100 μM ACh, was automatically applied via a custom-made magnetic valve system combined with a manifold mounted closely above the oocyte, thus allowing a fast (<300 ms) and reproducible solution exchange. Agonist pulses (2 s) were applied in 4-min intervals. Toxins were manually applied from a 10× stock solution in the 50-μL measuring chamber and preincubated for 3 min. Current responses were normalized to control responses before toxin application. GraphPad Prism (version 9.3.1) was used for data analysis, and a four-parameter logistic fit (Hill-fit) with plateaus constrained to 100% and 0% was used to generate dose–response curves. Oocytes from at least two frogs were used for each data point.Functional experiments were performed as previously described by Giribaldi et al. . Briefly


Piper eriopodon is one of the Piper species found in the Sierra Nevada de Santa Marta, and the species has been reported with different compositions of their essential oils (EO). In this study, the volatile fractions/essential oil (by HS-SPME/SDE/MWHD-GC–MS/1H-NMR) of different parts from the plant were characterized, and assessments of the in vitro bio-properties of the leaf EO were conducted. The results indicated the following: (i) in the volatile fractions were β-caryophyllene (~23%)/myrcene (~20%) (inflorescences) and β-caryophyllene (~43%)/β-selinene (~20%) (leaves) using HS-SPME; myrcene (~31%)/β-pinene (~23%) (inflorescences), gibbilimbol B (~60%) (fruits) and gibbilimbol B (~46%)/β-caryophyllene (~11%) (leaves) through SDE; (ii) leaf EO contained gibbilimbol B (~72%), confirmed with 1H-NMR; (iii) the cytotoxic values (µg/mL) in erythrocytes/lymphocytes/Hep-2 were HC50: 115 ± 3 (eryth.), LC50: 71 ± 4 (lymph.) and LC50: 33 ± 2 (cell-line); (iv) the antibacterial susceptibilities  were 22.5 ± 0.4–97 ± 4 (Staphylococcus aureus), 23 ± 2–77 ± 4 (Escherichia coli) and 17 ± 1–48 ± 3 (Listeria monocytogenes); (v) the TAA value was 2249 ± 130 mmol Trolox®/kg; (vi) the IC50 value was 13±1 µg/mL (AChE) with 20 ± 0–37 ± 6% repellency . Thus, the EO of P. eriopodon leaves from northern Colombia could be a promising species for sustainable exploitation in the future due to its outstanding bioactivities. Moreover, as an additional value, the in vitro biological prospecting of this EO was assessed according to cytotoxic (on human cell models), antibacterial (three strains), antiradical (ABTS+•) and repellency/AChE inhibition assays.Nevertheless, information on the composition of volatile fractions of the plant, as well as the effects of the leaf EO as a repellent/acetylcholinesterase inhibitor, antibacterial  C.DC., and the leaf EO was a yellowish liquid (at room temperature) which became solid at 4 °C; the EO yield was 0.08%.The botanical sample was identified as The constituents (>0.4%) that were positively identified (~91–100%) as volatile metabolites in the different parts of plant, based on the elution order of the total ion current (TIC), are included in P. eriopodon leaves and inflorescences, ethyl acetate extracts were obtained from these parts and analyzed using GC–MS; the chemical compositions are included in As an additional process to corroborate the presence of gibbilimbol B and other terpene constituents in the 1H-NMR was used for obtaining the characteristic profile of EO and confirming the presence of gibbilimbol B  were as follows: δ 7.03 (d), 6.74 (d), 5.88 (s), 5.46–5.37 (m), 5.30 (s), 5.10–4.82 (m), 4.01 (s), 3.75 (s), 3.64 (t), 3.30 (d), 2.89 (dd), 2.59 (t), 2.35–2.31 (m), 2.29–2.20 (m), 2.17–2.03 (m), 2.00–1.92 (m), 1.82–1.43 (m), 1.36–1.20 (m), 1.12–1.03 (m), 1.00–0.96 (m), 0.88 (t), 0.84–0.77 (m), 0.72 (s).The characteristic signals for the yellowish liquid (EO) via 16H24O, GC–MS -tR 80.62 min, m/z (%): 232.18 , 107.04 (100)] were as follows: 1H-δ 7.03 , 6.74 , 5.46-5.37 , 2.59 , 2.29-2.20 , 2.00-1.92 , 1.36-1.23 4–) and 0.88 . In addition, some unique/particular signals assigned to hydrogen atoms bonded to sp2 carbons of β-caryophyllene  were observed; thus, δ 5.10-4.82 , 4.01  and 3.75 .From previous signals, those corresponding to gibbilimbol B , whilst the effect of the different amounts of EO was significant  on each strain, all of the above evidenced by the trends shown in p: 0.1468-0.2394, F (3.18–5.35) < Fcrit (18.5–19)] with the control antibiotic (4–30 µg) when compared, indicating that the EO and control had similar antibacterial effectiveness under the conditions of this assay. In contrast, the other EO amounts tested (8–16 µg), pursuant to the Anova, were significantly different  > Fcrit (18.5)]) in relation to the standard antibiotic.A remarkable bioproperty demonstrated by Sitophilus zeamais (Coleoptera: Curculionidae) and the in vitro inhibition of the acetylcholinesterase enzyme (AChE) were also evaluated for this EO. As a result, the degree of repellency for the EO was moderate , and the inhibitory effect of EO on AChE was significant , although it did not exceed the value obtained for the positive control.Then, the repellent effect against ®/kg) of the EO evaluated, the reactivity of the EO towards the ABTS+• radical-cation, as a measure of its antioxidant capacity, was slightly higher (ratio 1.04) than that of the “control” antioxidant (BHA); however, there was not a significant difference  between these values, and therefore, the radical-scavenging capacity of P. eriopodon EO was comparable to the BHA.Finally, according to the TAA value , using HS-SPME [with DVB/CAR/PDMS (50/30 μm) as the fiber coating] and GC/MS, contained terpene hydrocarbons between ~20-86%, and in eight  of ten species, this type of compound family was predominant (~47–86%). The main terpenes identified were β-caryophyllene (8.5 ± 0.2-27.70 ± 0.08%), myrcene (16.06 ± 0.04–34.9 ± 0.4%) and ocimene (15.6 ± 0.4–31.5 ± 0.6%). Furthermore, Liu et al.  of the plant could be playing an ecological role , pollinator attractants . Once the EO was decanted and dehydrated, it was prepared for the spectroscopic analysis (GC–MS and 1H-NMR)  was carried out following the procedure described by Muñoz-Acevedo et al. [ABTSo et al. .p < 0.05, F < 0.05), the t-test ) and two-factor analysis of variance (Anova)  in Statistica software .Statistical treatment. The data obtained from the biological tests of the EO were treated with the corresponding figures of merit . In addition, the analysis tools used to determine the significance between the data were F-test two-sample for variance  in agreement to the established chemical compositions; (ii) based on the antibacterial efficacy of EO, it could be used as a food protectant/preservative, an attribute that would be reinforced by its anti-radical (antioxidant) capacity equivalent to BHA . In addition, the selective cytotoxicity (SI: ~2) of the EO on the Hep-2 line regarding lymphocytes would suggest its probable use as a chemotherapeutic agent (anticancer/anti-proliferative) against cervical adenocarcinoma. Lastly, despite the moderate degree of repellency and significant inhibition of AChE by EO, it could be applied as a bio-pesticide. (iii) As the main component found in the leaf EO was gibbilimbol B (~72% and probably responsible for the bioproperties), the final use/application could be carried out with the mixture (EO matrix) more easily than with the isolated alkenyl-phenol because the technique used (MWHD) would be much faster (1 h) and less tedious than the process with solvents (extraction/concentration/purification).From this research, the following could be concluded: (i) This is the first report of volatile fractions of the different parts of industry ), which