NONO promotes gallbladder cancer cell proliferation by enhancing oncogenic RNA splicing of DLG1 through interaction with IGF2BP3/RBM14.

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

The Potential of Vitamin K as a Regulatory Factor of Bone Metabolism-A Review.

Vitamin K (VK), a fat-soluble vitamin, is essential for the clotting of blood because of its role in the production of clotting factors in the liver. Moreover, researchers continue to explore the role of VK as an emerging novel bioactive molecule with the potential function of improving bone health. This review focuses on the effects of VK on bone health and related mechanisms, covering VK research history, homologous analogs, dietary sources, bioavailability, recommended intake, and deficiency. The information summarized here could contribute to the basic and clinical research on VK as a natural dietary additive and drug candidate for bone health. Future research is needed to extend the dietary VK database and explore the pharmacological safety of VK and factors affecting VK bioavailability to provide more support for the bone health benefits of VK through more clinical trials.

Resistance to the DMI fungicide mefentrifluconazole in Monilinia fructicola: risk assessment and resistance basis analysis.

BACKGROUND: Brown rot disease, caused by Monilinia fructicola, poses a significant challenge to peach production in China. The efficacy of mefentrifluconazole, a new triazole fungicide, in controlling brown rot in peaches has been remarkable. However, the resistance risk and mechanism associated with this fungicide remain unclear. This study was designed to assess the resistance risk of M. fructicola to mefentrifluconazole and reveal the potential resistance mechanism. RESULTS: The mean median effective concentration (EC50 ) of 101 M. fructicola isolates to mefentrifluconazole was 0.003 µg mL-1 , and the sensitivity exhibited a unimodal distribution. Seven mefentrifluconazole-resistant mutants were generated from three parental isolates in the laboratory through fungicide adaption. The biological characteristics of the resistant mutants revealed that three of them exhibited enhanced survival fitness compared to the parental isolates, whereas the remaining four mutants displayed reduced survival fitness. Mefentrifluconazole showed strong positive cross-resistance with fenbuconazole, whereas no cross-resistance was observed with pyrimethanil, procymidone or pydiflumetofen. No overexpression of MfCYP51 gene was detected in the resistant mutants. Multiple sequence alignment revealed that three resistant mutants (MXSB2-2, Mf12-1 and Mf12-2) had a point mutation (G461S) in MfCYP51 protein. Molecular docking techniques confirmed the contribution of this point mutation to mefentrifluconazole resistance. CONCLUSION: The risk of M. fructicola developing resistance to mefentrifluconazole is relatively low-to-medium and point mutation G461S in MfCYP51 could confer mefentrifluconazole resistance in M. fructicola. This study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for mefentrifluconazole. © 2023 Society of Chemical Industry.

Effects of Dietary Valine Chelated Zinc Supplementation on Growth Performance, Antioxidant Capacity, Immunity, and Intestine Health in Weaned Piglets.

This study was conducted to investigate the effects of dietary valine chelated zinc (ZnVal) supplementation on growth performance, antioxidant capacity, immunity, and intestine health in weaned piglets. A total of 240 healthy 35-day-old weaned piglets (Duroc × Landrace × Yorkshire, average weight 10.70 ± 0.14 kg) were randomly divided into five groups with six replicate pens and eight piglets per pen. Dietary treatments were a corn-soybean meal basal diet supplemented with 0, 25, 50, 75, and 100 mg/kg ZnVal, respectively. The experiment lasted for 28 days. Results showed that average daily gain (ADG) was increased (P < 0.05) by ZnVal with 75-100 mg/kg supplementation on days 15-28 and with 50-100 mg/kg supplementation on days 1-28. Supplementation of 25-100 mg/kg ZnVal reduced (P < 0.01) the diarrhea rate of weaned piglets on days 1 to 14 and 1 to 28. Dietary supplementation with 25-100 mg/kg ZnVal increased (P < 0.05) copper/zinc-superoxide dismutase (Cu/Zn-SOD) and decreased malonaldehyde (MDA) activities in the serum on day 14 and 28. Supplementation of 25-100 mg/kg ZnVal increased (P < 0.05) glutathione peroxidase (GSH-Px) activity in serum on day 14. Additionally, the supplementation of 75 mg/kg ZnVal significantly increased the activity of superoxide dismutase (SOD) and Cu/Zn-SOD in the liver (P < 0.05). Furthermore, the supplementation of 25-100 mg/kg ZnVal significantly increased the total antioxidant capacity (T-AOC) in the liver (P < 0.05). Higher (P < 0.05) concentrations of IgG in the serum were measured from piglets supplemented with 75-100 mg/kg ZnVal on day 14 and dietary supplementation with 25-100 mg/kg ZnVal increased the level of immunoglobulin G (IgG) in serum on day 28 (P < 0.05). In addition, higher (P < 0.05) concentrations of immunoglobulin A (IgA) in the duodenum and ileum were measured from piglets supplemented with 75 mg/kg ZnVal and the supplementation of 25-100 mg/kg ZnVal also showed a higher (P < 0.05) concentration of immunoglobulin G (IgG) in duodenum. Supplementation of 50-100 mg/kg ZnVal increased the villus height and villus height/crypt depth of jejunum (P < 0.05). Moreover, dietary supplementation with 75-100 mg/kg ZnVal showed a higher (P < 0.05) concentration of zinc in the liver and supplementation of 50-100 mg/kg ZnVal increased (P < 0.05) the concentration of zinc in the heart, spleen, and kidney. In conclusion, the present research showed that supplementation of ZnVal improves growth performance by increasing antioxidant capacity and immunity and regulating intestinal morphology and the optimal inclusion level of ZnVal was 65~80 mg/kg.

Retinoic acid enhances ovarian steroidogenesis by regulating granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway.

INTRODUCTION: Ovarian steroidogenesis not only affects the embryonic development and pregnancy outcome, but also associates with many diseases in mammals and women. Exploring the nutrients and mechanisms influencing ovarian steroidogenesis is critical to maintaining the optimal reproductive performance, as well as guaranteeing body health. OBJECTIVES: This research aimed to explore the effect of retinol metabolism on ovarian steroidogenesis and the underlying mechanisms. METHODS: Comparative transcriptomic analysis of ovaries from normal and low reproductive performance sows were performed to identify the main causes leading to low fertility. The metabolites regulating steroid hormones synthesis were investigated in ovarian granulosa cells. Gene interference, overexpression, dual-luciferase reporter assays, chromatin immunoprecipitation and transcriptome analysis were further conducted to explore the underlying mechanisms of Aldh1a1 mediating ovarian steroidogenesis. RESULTS: Transcriptome analysis of ovaries from normal and low reproductive performance sows showed the significant differences in both retinol metabolism and steroid hormones synthesis, indicating retinol metabolism probably influenced steroid hormones synthesis. The related metabolite retinoic acid was furtherly proven a highly active and potent substance strengthening estrogen and progesterone synthesis in ovarian granulosa cells. For the first time, we revealed that retinoic acid synthesis in porcine and human ovarian granulosa cells was dominated by Aldh1a1, and required the assistance of Aldh1a2. Importantly, we demonstrated that Aldh1a1 enhanced the proliferation of ovarian granulosa cells by activating PI3K-Akt-hedgehog signaling pathways. In addition, Aldh1a1 regulated the expression of transcription factor MESP2, which targeted the transcription of Star and Cyp11a1 through binding to corresponding promoter regions. CONCLUSION: Our data identified Aldh1a1 modulates ovarian steroidogenesis through enhancing granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway. These findings provide valuable clues for improving ovarian health in mammals.

Effects of vitamin K supplementation on reproductive performance and bone metabolism-related biochemical markers in lactation sows.

OBJECTIVE: This study was conducted to evaluate the effects of vitamin K (VK) supplementation on reproductive performance and bone metabolism-related biochemical markers in sows. METHODS: Twenty-four Large White×Landrace sows (mean parity 4.04) were randomly assigned to two dietary treatments (NC diet, a basal diet with 0.5 mg/kg of VK3; VK diet, a basal diet with 5 mg/kg of VK3) with twelve replicates per treatment and one sow per replicate according to parity. The experiment started on day 107 of gestation and lasted until day 21 of lactation (weaning). RESULTS: We observed that there were no differences (p>0.05) in average daily feed intake, backfat loss of sows, live piglet number at birth and weaning, average birth weight, average weaning weight, and average daily gain of piglets between two treatments. The apparent total tract digestibility of phosphorus was increased (p<0.05) in VK sows compared with NC sows. The serum bone alkaline phosphatase, osteocalcin, type I procollagen aminoterminal peptide, and type I procollagen carboxyl-terminal peptide on day of farrowing were higher (p<0.05) in VK sows than in NC sows. The serum phosphorus, parathyroid hormone, tartrate-resistant acid phosphatase, and tumor necrosis factor-alpha on day of weaning were lower (p<0.05) in VK sows compared with NC sows. CONCLUSION: Therefore, the overall results suggested that increasing dietary VK3 (0.5 to 5 mg/kg) during lactation improved the apparent total tract digestibility of phosphorus and serum bone metabolism biochemical markers in sows.

Supplementation with alpha-glycerol monolaurate during late gestation and lactation enhances sow performance, ameliorates milk composition, and improves growth of suckling piglets.

BACKGROUND: Physiological changes during lactation cause oxidative stress in sows, reduce immunity, and hamper the growth capacity of piglets. Alpha-glycerol monolaurate (α-GML) has potential for enhancing the antimicrobial activity of sows and the growth of suckling piglets. METHODS: Eighty sows were allocated randomly to four groups: basal diet and basal diets supplemented with 500, 1000, or 2000 mg/kg α-GML. The experiment started on d 85 of gestation and lasted until piglets were weaned on d 21 of lactation. The number of live-born piglets was standardized to 12 ± 1 per sow on day of parturition. On d 0 and 21 of lactation, body weight of piglets was measured and milk samples were obtained from sows, and serum samples and feces from piglets were obtained on d 21. RESULTS: Feed intake, backfat loss, and weaning estrus interval did not differ among the four groups of sows. Maternal α-GML supplementation increased (P < 0.05) the body weight of piglets at weaning and the apparent total tract digestibility of crude fat of sows. The immunoglobulin A and immunoglobulin G levels were greater (P < 0.05) in a quadratic manner in the milk of sows as dietary α-GML increased. Concerning fatty acid profile, C12:0, C15:0, C17:0, C18:2n6c, C18:3n3, C24:0, and C22:6n3 were higher (P < 0.05) in linear and quadratic manners in colostrum of sows-fed α-GML diets compared with the control sows. There was lower (P < 0.05) n-6:n-3 polyunsaturated fatty acid ratio in milk than in the control sows. Maternal α-GML increased the abundance of Firmicutes (P < 0.05) and decreased the abundance of Proteobacteria (P < 0.05) of piglet fecal microbiota. CONCLUSIONS: Dietary supplementation with α-GML improved milk immunoglobulins and altered fatty acids of sows, thereby improving the health of piglets.

Effects of a functional fatty acid blend on growth performance, intestinal morphology, and serum profiles in weaned piglets.

OBJECTIVE: The objective of this study was to determine whether dietary supplementation with a functional fatty acid blend (FA) that contains 31.4% butyric acid and 4.99% medium-chain FA improve growth performance, antioxidant capacity, immunity status, and anti-inflammatory ability in weaned piglets. METHODS: One hundred and forty-four healthy piglets (Duroc×Landrace×Yorkshire) with an average body weight (BW) of 7.98±3.43 kg were randomly divided into three groups with six replicate pens and eight piglets per pen: Normal control (NC): a corn-soybean basal diet; FA1: a basal diet supplemented with 1,000 mg/kg of a functional FA; FA2: a basal diet supplemented with 2,000 mg/kg of a functional FA. The experiment lasted for 28 d. On d 14 and 28, one piglet in each pen from NC and FA2 groups was randomly selected for antioxidative index and immunoglobulins. On d 28, one piglet in each pen from NC and FA2 groups was randomly selected for intestinal morphology and inflammatory factor. RESULTS: We observed that FA supplementation linearly increased (p<0.05) average daily gain and the final BW. There was higher (p<0.05) catalase on d 14, and immunoglobulin (Ig) A and IgM on d 28 in piglets supplemented with FA2 than in the NC group. Moreover, dietary FA2 reduced (p<0.05) crypt depth of ileum in piglets. The concentrations of tumor necrosis factor-α, interleukin (IL)-1ß, IL-8, and IL-10 in jejunum were lower (p<0.05) in the FA2 group compared with the NC group. CONCLUSION: Therefore, the overall results suggests that the FA may help to improve gut health, antioxidant status, and immune parameters resulting in the improvement of growth performance.

Effects of essential oil coated with glycerol monolaurate on growth performance, intestinal morphology, and serum profiles in weaned piglets.

OBJECTIVE: This study aimed to investigate the effects of essential oil coated with glycerol monolaurate (GML) on the growth performance, intestinal morphology, and serum profiles of weaned piglets. METHODS: A total of 144 weaned piglets (Duroc×[Landrace×Yorkshire], average weight 8.07±3.33 kg) were randomly assigned to three groups with six replicate pens and eight piglets per pen: i) CON: a corn-soybean basal diet; ii) LEG: with 1,000 mg/kg essential oil coated with GML; and iii) HEG: with 2,000 mg/kg essential oil coated with GML. RESULTS: Results showed that average daily gain was increased (p<0.05) linearly by essential oil coated with GML supplementation on day 14 to 28 and day 0 to 28 compared with the CON group. Dietary supplementation with HEG increased (p<0.05) total antioxidant capacity and catalase activity on day 14, and immunoglobulin A (IgA) and IgM concentration on day 28 and tended to increase IgG on day 28. In addition, the crypt depth in the jejunum was reduced (p<0.05), and villus height and villus height/crypt depth in the ileum were increased (p<0.05) in the HEG group compared with the CON group. Moreover, lower (p<0.05) concentrations of tumor necrosis factor-α, interferon-γ, interleukin-1ß (IL-1ß), IL-8, and IL-10 were observed in the jejunum of piglets supplemented with HEG compared with the CON group. In addition, dietary HEG tended to decrease IL-6 level in the jejunum of piglets compared with the CON group. CONCLUSION: Dietary essential oil coated with GML can improve growth performance of weaned piglets. Moreover, supplementing 2,000 mg/kg essential oil coated with GML was demonstrated to improve antioxidant ability, and intestinal morphology, and reduce jejunal inflammatory factor levels.

Vitamin K2 Improves Osteogenic Differentiation by Inhibiting STAT1 via the Bcl-6 and IL-6/JAK in C3H10 T1/2 Clone 8 Cells.

Osteogenic activity of vitamin K2 (VK2), a small molecular nutrient, has been suggested. However, the underlying mechanisms have not been fully elucidated. Therefore, this study aimed to explore the mechanisms by which VK2 promotes osteogenic differentiation. The effects of VK2 on osteogenic differentiation indicators were determined in C3H10 T1/2 clone 8 cells. The RNA-seq analysis was used to explore the hypothesis that VK2 promotes osteogenic differentiation. Small interfering RNA (siRNA) assay and plasmid transfection assay were used to determine the potential role of VK2 in the modulation of Bcl-6/STAT axis and IL-6/JAK/STAT signaling pathway. VK2 significantly increased alkaline phosphatase (ALP) activity, ALP, osteocalcin (OCN), and RUNX2 abundance, and RUNX2 protein expression. RNA-seq analysis showed that there were 314 differentially expressed genes (DEGs) upregulated and 1348 DEGs downregulated by VK2. PPI analysis determined the top 10 hub genes upregulated or downregulated by VK2. Overexpression of Bcl-6 increased osteogenic differentiation and decreased expression of STAT1. Administration with VK2 restored the inhibition by siBcl-6 in osteogenic differentiation. Knockdown of IL-6 decreased the mRNA levels of genes associated with the JAK/STAT signaling pathway, and increased markers of osteoblast differentiation. Furthermore, treatment with VK2 improved inhibition in osteogenic differentiation and decreased enhancement of JAK/STAT signaling pathway related genes by overexpression of IL-6. Our study suggests that VK2 could improve osteogenic differentiation via the Bcl-6/STAT axis and IL-6/JAK/STAT signaling pathway.

Effects of the methionine hydroxyl analog chelated microminerals on growth performance, antioxidant status, and immune response of growing-finishing pigs.

This study aimed to investigate the effects of methionine hydroxyl analog chelated microminerals (MHA-M) replacing inorganic microminerals (ITMs) on the growth performance, fecal microminerals concentrations, immune function, and antioxidant status in growing-finishing pigs; 253 pigs (average 33.68 kg body weight) were randomly assigned to six treatments with six replicates each treatment: (1) ITM: a basal diet with Cu, Fe, Mn, and Zn from sulfates providing 20, 100, 40, and 60 mg/kg; (2-6): 1/5MHA-M, 2/5MHA-M, 3/5MHA-M, 4/5MHA-M, and MHA-M was replaced with 20%, 40%, 60%, 80%, and 100% MHA-M. Results showed that the average daily gain (ADG) in the 1/5MHA-M and 2/5MHA-M was greater than other groups in the whole period. Fecal Cu, Fe, Mn, and Zn concentrations had decreased as the intake of trace minerals decreases. The ITM group decreased the fecal Zn concentration on Days 35, 70, and 91, and Fe concentration on Day 70, and increased the Mn concentration on Day 70 compared with MHA-M group. Pigs fed 1/5MHA-M, 2/5MHA-M, and MHA-M had a higher immune function and antioxidant status in serum compared with ITM, 3/5MHA-M, and 4/5MHA-M on Day 35. In conclusion, treatment with 1/5MHA-M and 2/5MHA-M could reduce the excretion of fecal microminerals and improve the immune function and antioxidant capacity compared with the ITM group.

Effects of Pelleting and Long-Term High-Temperature Stabilization on Vitamin Retention in Swine Feed.

The objective of this study was to study the effect of pelleting and long-term high-temperature stabilization on the retention of vitamin A, vitamin E, vitamin B2, and vitamin B6 in swine feed. Piglet diets (diet 1 and 3) were pelleted after conditioning at 83 °C for 120 s, and were high-temperature stabilized at 90 °C for 8.5 min after pelleting; the finishing pig diets (diet 2, 4, and 5) were pelleted after conditioning at 82 °C for 90 s, and were high-temperature stabilized at 85 °C for 9 min after pelleting; the samples were obtained before condition, after condition, after pelleting, and after cooling. The contents of vitamin A and vitamin E in diets 1−5 and vitamin B2, and vitamin B6 in diets 3−5 were detected. The results showed that: (1) the conditioning process had no significant effect on the retention of vitamin A, vitamin E, vitamin B2, and vitamin B6 in all experimental diets (p > 0.05); (2) the pelleting process and high-temperature stabilization process after pelleting had different degrees of influence on vitamins, among which the stabilization process had a more significant effect on the retention of vitamins. After pelleting and long-term high-temperature stabilization, the retention of vitamin A, vitamin E, and B2, and vitamin B6 were 68.8−77.3%, 56.9−90.1%, 63.8−70.3%, and 60.1−67.0%, respectively. In the process of pelleting and long-term high-temperature stabilization, the retention of vitamin A, vitamin E, vitamin B2, and vitamin B6 in the feed were significantly reduced (p < 0.05). Therefore, vitamin loss during high temperature and over a long period of time is worth considering, and vitamins must be over-supplemented.

Effects of α-glycerol monolaurate on intestinal morphology, nutrient digestibility, serum profiles, and gut microbiota in weaned piglets.

This experiment was conducted to investigate the effects of dietary supplementation of α-glycerol monolaurate (α-GML) on the growth performance, nutrient digestibility, serum profiles, intestinal morphology, and gut microbiota of weaned piglets. A total of 96 healthy 28-d-old (Duroc × Landrace × Yorkshire) weaned piglets with body weight of 8.34 ± 0.05 kg were randomly divided into 2 treatment groups with 6 replicate pens and 8 piglets per pen. The control group was fed a basal diet and the experimental group was fed the basal diet supplemented with 1,000 mg/kg α-GML. The experiment lasted for 28 d. Dietary supplementation with α-GML had no effect on average daily gain, average daily feed intake, or gain to feed ratio in piglets (P > 0.05); however, it reduced (P < 0.05) diarrhea rate of piglets on days 15 to 28. The apparent total tract digestibility of dry matter (DM), crude protein (CP), ether extract (EE), and gross energy (GE) on day 14, and DM, organic matter, CP, EE, and GE on day 28 increased (P < 0.05) with α-GML supplementation. Moreover, higher (P < 0.05) glutathione peroxidase activity and interleukin-10 (IL-10) concentration, and lower (P < 0.05) malondialdehyde and tumor necrosis factor-α concentrations were observed in piglets supplemented with α-GML compared with the control group on day 14. Compared with the control group, the villus height/crypt depth in the duodenum and villus height in the jejunum and ileum were significantly greater (P < 0.05) in the α-GML group. Dietary α-GML supplementation significantly increased (P < 0.05) the relative abundance of Firmicutes, while decreasing (P < 0.05) Bacteroidota and Campilobacterota in the cecal contents; significantly increased (P < 0.05) the relative proportion of Lactobacillus and Blautia species, reduced (P < 0.05) Eubacterium_rectale_ATCC_33656, Campylobacter, and uncultured_bacterium_Alloprevotella species. Thus, dietary α-GML supplementation at 1,000 mg/kg reduces diarrhea rate, improves intestinal morphology, nutrient digestibility, antioxidant capacity, and immune status, and ameliorates gut microbiota in weaned piglets.

Glycerol monolaurate (GML) is naturally present in breast milk as well as other natural sources such as coconut oil and is widely used as a food additive. Dietary α-GML is used in animal production due to its safe-guarding health and growth-promoting effects. In the present study, α-GML was evaluated for growth performance, blood parameters, and intestinal health in piglets. Dietary α-GML helped piglets digest dry matter, crude protein, ether extract, and gross energy in feed. The blood parameters and intestinal structure of piglets fed the diet containing 1,000 mg/kg α-GML were improved. In addition, α-GML supplementation promoted the colonization of beneficial bacteria and inhibited the number of harmful bacteria. In the current study, dietary α-GML was responsible for improving the health status, intestinal morphology, and digestion and absorption of nutrients of weaned piglets with less diarrhea.

Menaquinone 4 Reduces Bone Loss in Ovariectomized Mice through Dual Regulation of Bone Remodeling.

Epidemiologic studies showed that higher vitamin K (VK) consumption correlates with a reduced risk of osteoporosis, yet the dispute remains about whether VK is effective in improving bone mineral density (BMD). We sought to discover the anti-osteoporotic effect of menaquinone-4 (MK-4) and evaluate the expression of critical genes related to bone formation and bone resorption pathways in the body. Fifty female C57BL/6 mice (aged 13 weeks) were randomly arranged to a sham-operated group (SHAM, treated with corn oil) and four ovariectomized groups that were administered corn oil (OVX group), estradiol valerate (EV, 2 mg/kg body weight as the positive control), low or high doses of VK (LVK and HVK; 20 and 40 mg MK-4/kg body weight, respectively) by gavage every other day for 12 weeks. Body and uterine weight, serum biochemical indicators, bone microarchitecture, hematoxylin-eosin (HE) staining, and the mRNA expression of critical genes related to bone formation and bone resorption pathways were assessed. Either dose of MK-4 supplementation increased the alkaline phosphatase (ALP), decreased the undercarboxylated osteocalcin (ucOC) and tartrate-resistant acid phosphatase (TRACP, p < 0.05) levels, and presented higher BMD, percent bone volume (BV/TV), trabecular thickness (Tb.Th), and lower trabecular separation (Tb.Sp) and structure model index (SMI, p < 0.05) compared with the OVX group. Additionally, both doses of MK4 increased the mRNA expression of Runx2 and Bmp2 (p < 0.05), whereas the doses down-regulated Pu.1 and Nfatc1 (p < 0.05) mRNA expression, the high dose decreased Osx and Tgfb (p < 0.05) mRNA expression, and the low dose decreased Mitd and Akt1 (p < 0.05) mRNA expression. These data show the dual regulatory effects of MK-4 on bone remodeling in ovariectomized mice: the promotion of bone anabolic activity and inhibition of osteoclast differentiation, which provides a novel idea for treating osteoporosis.

Effects of Sources and Forms of Vitamin K3 on Its Storage Stability in Vitamin Premixes or Vitamin Trace Mineral Premixes.

Six types of vitamin K3 (VK3); two sources (menadione sodium bisulfite, MSB; menadione nicotinamide bisulfite, MNB), and three different forms (crystal, micro-capsule, and micro-sphere) were used to determine the retention of VK3 in vitamin premixes (Experiment 1) or vitamin trace mineral (VTM) premixes (Experiment 2) after 1, 2, 3, and 6 months of storage. The retention of VK3 in vitamin premixes was evaluated at 25 °C/60% relative humidity or 40 °C/75% relative humidity in an incubator in Experiment 1 and in VTM premixes (choline chloride: 0 vs. 16,000 mg/kg) stored at room temperature in Experiment 2. The VK3 retention in vitamin premix or VTM premix decreased significantly with the extension of storage time (p < 0.05). In Experiment 1, the VK3 retention was higher in the 25 °C/60% incubator (56%) than in the 40 °C/75% incubator (28%). The MNB retention (52%) was higher than MSB retention (32%). The retention of VK3 in micro-capsules (43%) or micro-spheres (48%) was higher than the crystal form (35%) after six months of storage. In Experiment 2, there was no difference between the retention of MSB (49%) or MNB (47%). The retention of VK3 of micro-capsule (51%) or micro-sphere (54%) was higher than that of crystal form (40%). The VK3 retention was higher in the choline-free group (51%) than in the choline group (47%) after six months of storage. Finally, the predicted equations of VK3 retention with storage time in vitamin premixes or VTM premixes were established. The R2 of the prediction equations was ≥0.9005, indicating that time is an important factor in predicting VK3 retention. In conclusion, the higher temperature-relative humidity, choline had negative effects on VK3 retention during premix storage. MNB retention was higher than MSB during storage of vitamin premix. The encapsulated forms of VK3, micro-capsules and micro-spheres, could improve VK3 storage stability in vitamin premix and VTM premix.

Effects of Sources or Formulations of Vitamin K3 on Its Stability during Extrusion or Pelleting in Swine Feed.

Two studies were conducted to determine the stability of vitamin K3 (VK3) in swine diets during extrusion or pelleting. The two sources were menadione sodium bisulfite (MSB) and menadione nicotinamide bisulfite (MNB), and the three formulations were crystal micro-capsule formulation and micro-sphere formulation. The recovery of six types of VK3 in swine diets was investigated after extrusion at 100 °C or 135 °C in Experiment 1. The recovery of six types of VK3 was investigated when the diets were pelleted at 60 °C (low temperature; LT) or 80 °C (high temperature; HT) and the length to diameter ratios were 5.2:1 (low length to diameter ratio; LR) or 7.2:1 (high length to diameter ratio; HR) in Experiment 2. In Experiment 1, MNB recovery (72.74%) was higher than MSB recovery (64.67%) after extrusion, while recovery of VK3 of crystal (74.16%) was higher than the recovery of micro-capsule (65.25%) and micro-sphere (66.72%). The recovery of VK3 (70.88%) was higher when extruded at 100 °C than that at 135 °C (66.54%). In Experiment 2, MNB recovery (86.21%) was higher than MSB recovery (75.49%) after pelleting, while the recovery of VK3 of micro-capsule (85.06%) was higher than the recovery of crystal (81.40%) and micro-sphere (76.09%). The recovery of VK3 (75.50%) was lower after HTHR pelleting than LTLR (83.62%), LTHR (81.52%) or HTLR (82.76%) treatment. Our results show that MNB has greater stability than MSB. VK3 of crystal or VK3 of micro-capsule were recommended for extrusion or pelleting, respectively.

Long noncoding RNA Linc00337 functions as an E2F1 co-activator and promotes cell proliferation in pancreatic ductal adenocarcinoma.

BACKGROUND: Long noncoding RNA (lncRNA) Linc00337 has been implicated in lung, gastric, colorectal and esophageal squamous cell carcinoma progression via various mechanisms; however, its clinicopathological significance and role in pancreatic ductal adenocarcinoma (PDAC) progression remains largely unknown. METHODS: Multiple approaches such as bioinformatic analysis, Transfection, quantitative real-time-PCR, Western blotting, animal studies, RNA-immunoprecipitation (RIP), RNA-pulldown and RNA-Fluorescence in situ hybridization (RNA-FISH) and were utilized to explore the role of Linc00337 in PDAC. RESULTS: Here we identified Linc00337 is an oncogenic lncRNA during PDAC progression. We found that the expression of Linc00337 is elevated in PDAC tissues and the higher Linc00337 predicts dismal prognosis. Functionally, Linc00337 promotes PDAC cell proliferation and cell cycle transition both in vitro and in vivo. Mechanistically, Linc00337 binds to E2F1 and functions as an E2F1 coactivator to trigger the targets expression during PDAC progression. CONCLUSION: Our results demonstrate a reciprocal regulation mechanism between Linc00337 and E2F1 in PDAC progression and report the clinical value of Linc00337 for PDAC prognosis and treatment.

Evaluation of Extrusion Temperatures, Pelleting Parameters, and Vitamin Forms on Vitamin Stability in Feed.

Two experiments were conducted to determine the stability of microencapsulated and non-microencapsulated forms of vitamins in diets during extrusion and pelleting. We investigated the recovery of vitamins in swine diets after extrusion at 100 °C, 140 °C, or 180 °C. Next, two diets were conditioned at 65 °C (low temperature; LT) or 85 °C (high temperature; HT), and pellets were formed using a 2.5 × 15.0 mm (low length-to-diameter ratio; LR) or 2.5 × 20.0 mm (high length-to-diameter ratio; HR) die. The extrusion temperature had a significant effect on the recovery of vitamins E, B1, B2, B3, and B5 in the diets. The diet extruded at 100 °C had higher B1, B2, B3, and B5 vitamin recoveries than diets extruded at 140 °C and 180 °C. Microencapsulated vitamins A and K3 had greater stability than non-microencapsulated vitamins A and K3 at 100 °C and 140 °C extrusion. In the diet extruded at 180 °C, microencapsulated vitamins A, D3, and K3 had higher recoveries than non-microencapsulated vitamins A, D3, and K3. The recovery of vitamin K3 in diets after LTLR (low temperature + low length-to-diameter ratio) or HTLR (high temperature + low length-to-diameter ratio) pelleting was greater (p < 0.05) than after LTHR (low temperature + high length-to-diameter ratio) and HTHR (high temperature + high length-to-diameter ratio) pelleting. Our results clearly show that low extrusion temperature and low pellet temperature, and a low length-to-diameter ratio (L:D ratio) for pellet mill die are recommended for pig feed. Moreover, microencapsulated vitamins had greater stability compared to non-microencapsulated vitamins.

Effects of Choline Chloride, Copper Sulfate and Zinc Oxide on Long-Term Stabilization of Microencapsulated Vitamins in Premixes for Weanling Piglets.

Two in vitro experiments were conducted to investigate the effects of choline chloride, copper sulfate (CuSO4) and zinc oxide (ZnO) on the stability of vitamin A (VA), vitamin D3 (VD3), vitamin E (VE), vitamin K3 (VK3), vitamin B1 (VB1), vitamin B2 (VB2), vitamin B6 (VB6), niacin, and pantothenic acid in vitamin and vitamin/trace mineral (VTM) premixes for weanling piglets after 0, 1, 2, 3, 6 and 12 months of premix storage. We developed predicted equations to estimate vitamin retention during storage. Two vitamin premixes (with or without choline) were formulated and stored at 25 °C and 60% humidity to establish the storage stability of vitamin premixes. Additionally, four VTM premixes were used to evaluate the effect of choline chloride (0 vs. 40,000 mg/kg) and trace minerals (Low CuSO4 + ZnO vs. High CuSO4 + ZnO) on vitamin stability in VTM premixes stored at room temperature (22 °C). In general, as storage time increased, residual vitamin activity decreased (p < 0.05). The results confirmed that VD3, VE, VB2, VB6, niacin and pantothenic acid were highly stable during storage, while the retention of VA, VK3 and VB1 was significantly affected by storage time and the presence of choline and high concentrations of Cu and Zn in the premix. After one year of storage, the retention of VE, VB2, VB6 niacin, and pantothenic acid was more than 90% in vitamin and VTM premixes. The retention of VD3 was more than 90% in vitamin premixes and more than 80% in VTM premixes after one year of storage. We conclude that current microencapsulation techniques for vitamin premixes appear to be inadequate to guarantee VA, VK3, and VB1 concentrations in VTM premixes.

Rapamycin Attenuates Splenomegaly in both Intrahepatic and Prehepatic Portal Hypertensive Rats by Blocking mTOR Signaling Pathway.

BACKGROUND: Spleen enlargement is often detected in patients with liver cirrhosis, but the precise pathogenetic mechanisms behind the phenomenon have not been clearly elucidated. We investigated the pathogenetic mechanisms of splenomegaly in both portal hypertensive patients and rats, and tried to identify the possible therapy for this disease. METHODS: Spleen samples were collected from portal hypertensive patients after splenectomy. Rat models of portal hypertension were induced by common bile duct ligation and partial portal vein ligation. Spleen samples from patients and rats were used to study the characteristics of splenomegaly by histological, immunohistochemical, and western blot analyses. Rapamycin or vehicle was administered to rats to determine the contribution of mTOR signaling pathway in the development of splenomegaly. RESULTS: We found that not only spleen congestion, but also increasing angiogenesis, fibrogenesis, inflammation and proliferation of splenic lymphoid tissue contributed to the development of splenomegaly in portal hypertensive patients and rats. Intriguingly, splenomegaly developed time-dependently in portal hypertensive rat that accompanied with progressive activation of mTOR signaling pathway. mTOR blockade by rapamycin profoundly ameliorated splenomegaly by limiting lymphocytes proliferation, angiogenesis, fibrogenesis and inflammation as well as decreasing portal pressure. CONCLUSIONS: This study provides compelling evidence indicating that mTOR signaling activation pathway plays a key role in the pathogenesis of splenomegaly in both portal hypertensive patients and rats. Therapeutic intervention targeting mTOR could be a promising strategy for patients with portal hypertension and splenomegaly.