Evaluation of the efficacy of a novel phytase in short-term digestibility and long-term egg production studies with laying hens.

Three independent trials were conducted to evaluate the efficacy of a novel phytase in laying hens. Trial 1 used a total of 90 laying hens (Lohmann Brown, 33-wk-old) fed either a negative control (NC) diet with 0.09% non-phytate P (NPP) or NC supplemented with 187.5 or 375 FYT phytase/kg feed for 4 d before collection of excreta and ileal digesta to measure ileal digestibility and retention of Ca and P. In trial 2 and 3, a total of 108 laying hens (Hy Line Brown, 25-wk-old) and 360 hens (Lohman Brown, 25-wk-old) were used, respectively. In both trials, the hens were randomly assigned to 3 dietary treatments: NC with 0.1% NPP, positive control (PC) and NC plus 187.5 FYT phytase/kg feed, the experimental diets were fed for 12 wk, and egg production and bone mineralization were measured. The results showed that the ileal digestibility of P increased both linearly (P = 0.012) and quadratically (P = 0.01) with increasing supplementation of phytase in trial 1. In trial 2, phytase supplementation significantly improved egg production, egg weight, and feed conversion ratio and reduced the percentage of broken eggs during the overall trial duration compared with NC. In trial 3, phytase significantly improved egg production, egg weight, and feed intake and reduced the percentage of broken eggs during the entire trial duration. In addition, percentage and weight of bone Ca and P increased significantly with added phytase. In trial 2 and 3, there was no significant difference between PC and the phytase treatment. In conclusion, the novel phytase significantly increased the ileal digestibility of P in a short-term digestibility study and improved egg production and bone mineralization in a 12-wk laying cycle. Ileal digestibility of P rather than P retention in short-term digestibility studies as well as egg production and whole tibia mineralization in long-term studies should be measured to demonstrate the efficacy of phytase in laying hens.

[Research of SIRT1 on promoting the proliferation, migration and lipid metabolism of nasopharyngeal carcinoma].

Objective: To analyze the differential expression of silent information regulator transcript-1 (SIRT1) in tissues and cells of nasopharyngeal carcinoma (NPC), to explore the effects of SIRT1 on the proliferation and migration of NPC cells, as well as the effects on and mechanisms of lipid metabolism in NPC cells. Methods: Experimental subjects: In this study, tissue specimens were obtained from patients who visited the Department of Otolaryngology and performed nasopharyngeal tissue biopsy in the Affiliated Hospital of Nantong University from 2019 to 2020. Among them, 6 cases were male, 6 cases were female, age range: 27-72 years old, including 7 cases of NPC diagnosed by pathology and 5 cases of normal nasopharyngeal mucosa. Experimental methods and outcome measures: Western Blot and quantitative real time polymerase chain reaction (qRT-PCR) were used to detect the protein and mRNA levels of SIRT1. CNE2 cell line was selected for subsequent experiments. Cell viability and migratory ability were evaluated by CCK8, wound healing and Transwell assays respectively. Animal xenograft tumor model was used to explore the role of SIRT1 inhibitor Ex527 on tumor growth in nude mice. Oil red and Bodipy were used to stain intracellular lipids. For the mechanical investigation, the interactions between SIRT1 and hypoxia inducible factor-1α (HIF-1α) were analyzed by immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP). Finally, statistical analysis was performed by SPSS 26.0 software, P<0.05 was considered statistically significant. Results: The levels of SIRT1 protein (1.005±0.168) and mRNA (5.829±2.395) in NPC tissues were higher than those in normal nasopharyngeal mucosa (0.181±0.042,1.995±1.605). Differences were statistically significant (t values were 6.438 and 2.759, both P<0.05). The mRNA and protein levels of CNE1, CNE2, 5-8F and 6-10B cell lines were also higher than those in normal nasopharynx epithelial cell line NP69. Besides, overexpression of SIRT1 correlated with the proliferation and migration of NPC cells. The tumorigenesis ability of nude mice in the Ex527 group was lower than that in the control group. The low SIRT1 expression reduced the protein level of the key enzymes of liposynthesis in NPC cells, improved the expression of lipolysis enzymes, while HIF-1α overexpression promoted lipid synthesis enzymes in NPC cells. SIRT1 inhibited HIF-1α transcription by enhancing deacetylation levels. The binding ability of HIF-1α to SIRT1 promoter regions decreased when NPC cells were hypoxic. Conclusions: SIRT1 promotes the proliferation, migration and lipid metabolism of nasopharyngeal carcinoma cells, which might be expected to provide new theoretical basis for prognosis judgment and gene therapy.

Research Note: Delay in sampling influences the profile of phytate in gizzard digesta and ileal digestibility of phosphorus in broilers.

The objective of this study was to investigate the effect of different durations of time delay when sampling digesta from the gizzard and ileum of broilers on the degradation of myo-inositol hexakisphosphate (InsP6) and digestibility of phosphorus (P). There was 1 experimental diet with a supplemental phytase activity of 1,212 phytase units/kg feed, which was provided to birds from day 13 to 18 after hatching. The diet was formulated to provide 6.6 g/kg Ca and 1.9 g/kg nonphytate P and fed to 24 cages of 6 birds. The 24 cages of birds were further randomly divided into 6 subgroups of 4 cages from which the digesta samples in the gizzard and ileum were collected at 0, 5, 10, or 20 min postmortem. The results showed that the concentration of InsP6 decreased linearly (P = 0.002), InsP5 decreased quadratically (P = 0.038), and the summation of concentrations of P in InsP6-4 decreased linearly (P = 0.028) in the gizzard digesta with the increasing delay of sampling. In the ileum, the digestibility of phytate P tended to decrease linearly (P = 0.087), and the digestibility of total P decreased linearly (P = 0.026) with prolonged delay. In conclusion, delay in sampling could alter the measured profile of InsP esters in gizzard digesta probably because of a continued effect of supplemental phytase, while the ileal digestibility of total P could diminish. Therefore, standard sampling procedures should be implemented to minimize variance.

Dietary sources of phosphorus affect postileal phosphorus digestion in growing pigs.

The present study was conducted to investigate the postileal P digestion response of growing pigs to dietary P concentrations and sources. Twenty-four pigs (57.3 ± 1.9 kg initial BW) fitted with T-cannulas at the distal ileum were assigned to a duplicated 12 × 4 incomplete Latin square design with 12 diets and 4 periods. The 12 experimental diets consisted of 9 cornstarch-based diets using soybean meal (SBM), corn distiller's dried grains with solubles (DDGS), and canola meal (CM) as assay ingredients at 3 levels and 3 corn-SBM-based practical diets with 3 levels of dicalcium phosphate (DCP). Chromic oxide was included as an indigestible marker to calculate apparent ileal digestibility (AID), apparent total tract digestibility (ATTD), and apparent postileal digestibility (APID) of P. Each period consisted of 5 d of adaption period, 2 d of fecal samples collection, and 2 d of ileal digesta collection. The results showed that ileal P output, fecal P output, ileal digested P, total tract digested P, and the AID and ATTD of P were affected by the interaction of P concentrations and sources ( < 0.01). When pigs were fed the semipurified diets containing SBM and corn-SBM-based practical diets, the AID and ATTD of P increased with increasing dietary P level (linear, < 0.01). However, there were no linear or quadratic responses in the AID and ATTD of P for pigs fed diets containing increasing levels of CM and corn DDGS. Postileal digested P, the proportion of ileal digested P to total tract digested P, the proportion of postileal digested P to total tract digested P, APID of P, and hindgut disappearance of P were affected by dietary sources of P ( < 0.01). When pigs were fed the semipurified diets containing SBM and CM, there were no differences between the AID and ATTD of P. In contrast, the ATTD of P were greater than the AID of P for pigs fed diets containing corn DDGS and corn-SBM-based practical diets with DCP supplementation. In summary, postileal P digestion response of growing pigs was affected by dietary P sources.

Effect of stachyose supplementation on growth performance, nutrient digestibility and caecal fermentation characteristics in broilers.

1. The objective of this study was to evaluate whether the oligosaccharide stachyose enhances gastrointestinal tract health by fermentation and proliferation of desirable bacteria species and thus affects growth performance and nutrient digestibility in broilers. 2. A total of 432 1-d-old male Arbor Acres (AA) broilers were randomly allocated to one of 6 treatments, with 12 replicate pens per treatment and 6 birds per pen. Chicks were fed a maize-hamlet protein 300 (HP300) basal diet with 0, 4.0, 8.0, 12.0 or 16.0 g/kg stachyose. A sixth diet contained no HP300 but soybean meal (SBM) and provided 8.7 g/kg stachyose and 3.1 g/kg raffinose. The duration of the study was 42 d. 3. Stachyose contents above 12.0 g/kg depressed group body weights, average daily gain and feed/gain but not feed intake during the whole experimental period. Broiler growth decreased linearly and quadratically with increasing stachyose content. No differences were detected between diets supplemented with 12.0 g/kg stachyose and SBM. 4. Nutrient digestibility tended to decrease but not significantly with increasing stachyose. 5. Stachyose content had no significant positive effects on caecal pH, microflora population and the resulting short-chain fatty acid (SCFA) metabolites during the 42 d experiment, with only butyrate differing significantly in the initial period.

Inhibition of GPI phospholipase C from Trypanosoma brucei by fluoro-inositol dodecylphosphonates.

Glycosyl phosphatidylinositol phospholipase C (GPI-PLC) of Trypanosoma brucei is inhibited by myo-inositol(Ins)-1-O-dodecylphosphonate (VP-602L). Several novel fluoro-substituted analogs of 2-deoxy-myo-Ins-1-O-dedecylphosphonate, among which 2-deoxy-2-fluoro-scyllo-Ins-1-O-dodecylphosphonate (VP-616L) was the most powerful, were shown to be competitive inhibitors of GPI-PLC. VP-616L was 14-fold more active than VP-602L. 2-Deoxy-2-fluoro-myo-Ins-1-O-dodecylphosphonate and 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate were 1.55- and 4.67-fold, respectively, more potent than VP-602L. Methyl 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate did not inhibit GPI-PLC. These observations provide several insights into how GPI-PLC might interact with its substrate at the active site. We surmise that (i) the 2-OH of Ins is probably dispensable for substrate recognition; (ii) an equatorially oriented active site residue might interact with substituents at the 2-position of Ins, and (iii) the negative charge on the phosphoryl at the 1-OH position of Ins might be important for substrate recognition.

Phosphatidylinositol phospholipase C is activated allosterically by the aminoglycoside G418. 2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecylphosphonate and its analogs inhibit glycosylphosphatidylinositol phospholipase C.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus is inhibited by myo-inositol-1-O-dodecylphosphonate (Ins-1-O-dodecylphosphonate) (Morris, J. C., Ping-Sheng, L., Shen, T. Y., and Mensa-Wilmot, K.(1995) J. Biol. Chem. 270, 2517-2524). A set of novel fluorinated 2-deoxy-Ins-1-O-dodecylphosphonates were tested against PI-PLC, with potent competitive inhibition by 2-deoxy-2-fluoro-scyllo-Ins-1-O-dodecylphosphonate (VP-616L) (Xi(50) = 0.09). 2-Deoxy-2-fluoro-myo-Ins-1-O-dodecylphosphonate and 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate were 8.3-fold and 4.8-fold less effective, respectively, than VP-616L. Methyl 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate was inactive. Also, a hundredfold less PI-PLC is required to cleave a glycosylphosphatidylinositol (GPI) than is needed to cleave PI. Implied in these observations are the following: (i) in powerful inhibitors an active site residue probably interacts with the equatorially oriented fluoro substituent; (ii) substrate recognition requires a negative charge on the phosphoryl at the Ins-1 position, and (iii) a GPI is better substrate than PI, for PI-PLC. Aminoglycoside antibiotics kanamycin A, gentamycin, and G418 stimulated PI-PLC cleavage of the GPI anchor of variant surface glycoprotein (VSG) from Trypanosoma brucei 2- to 4-fold. G418, which appears to act on the enzyme.substrate complex, increased kcat and Km 6.4-fold and 9.9-fold, respectively. PI-PLC was activated by G418 even in the presence of the inhibitor VP-616L. In control experiments, the lectin concanavalin A (ConA), which probably acts by substrate sequestration, inhibited both PI-PLC (Xi(50) = 0.00025) and GPI-specific phospholipase D (Xi(50) = 0.00018). G418 failed to activate PI-PLC when ConA was present. These observations indicate that G418 is an allosteric activator of Bacillus cereus PI-PLC. Since G418 stimulates a purified enzyme that is not involved in aminoglycoside metabolism, we propose that binding of aminoglycosides to cellular proteins could contribute to the development of the nephrotoxicity associated with the use of these aminoglycoside antibiotics.