Lysine Distinctively Manipulates Myogenic Regulatory Factors and Wnt/Ca2+ Pathway in Slow and Fast Muscles, and Their Satellite Cells of Postnatal Piglets.

Muscle regeneration, representing an essential homeostatic process, relies mainly on the myogenic progress of resident satellite cells, and it is modulated by multiple physical and nutritional factors. Here, we investigated how myogenic differentiation-related factors and pathways respond to the first limiting amino acid lysine (Lys) in the fast and slow muscles, and their satellite cells (SCs), of swine. Thirty 28-day-old weaned piglets with similar body weights were subjected to three diet regimens: control group (d 0-28: 1.31% Lys, n = 12), Lys-deficient group (d 0-28: 0.83% Lys, n = 12), and Lys rescue group (d 0-14: 0.83% Lys; d 15-28: 1.31% Lys, n = 6). Pigs on d 15 and 29 were selectively slaughtered for muscular parameters evaluation. Satellite cells isolated from fast (semimembranosus) and slow (semitendinosus) muscles were also selected to investigate differentiation ability variations. We found Lys deficiency significantly hindered muscle development in both fast and slow muscles via the distinct manipulation of myogenic regulatory factors and the Wnt/Ca2+ pathway. In the SC model, Lys deficiency suppressed the Wnt/Ca2+ pathways and myosin heavy chain, myogenin, and myogenic regulatory factor 4 in slow muscle SCs but stimulated them in fast muscle SCs. When sufficient Lys was attained, the fast muscle-derived SCs Wnt/Ca2+ pathway (protein kinase C, calcineurin, calcium/calmodulin-dependent protein kinase II, and nuclear factor of activated T cells 1) was repressed, while the Wnt/Ca2+ pathway of its counterpart was stimulated to further the myogenic differentiation. Lys potentially manipulates the differentiation of porcine slow and fast muscle myofibers via the Wnt/Ca2+ pathway in opposite trends.

Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae.

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

A chemical proteomics approach for global mapping of functional lysines on cell surface of living cell.

Cell surface proteins are responsible for many crucial physiological roles, and they are also the major category of drug targets as the majority of therapeutics target membrane proteins on the surface of cells to alter cellular signaling. Despite its great significance, ligand discovery against membrane proteins has posed a great challenge mainly due to the special property of their natural habitat. Here, we design a new chemical proteomic probe OPA-S-S-alkyne that can efficiently and selectively target the lysines exposed on the cell surface and develop a chemical proteomics strategy for global analysis of surface functionality (GASF) in living cells. In total, we quantified 2639 cell surface lysines in Hela cell and several hundred residues with high reactivity were discovered, which represents the largest dataset of surface functional lysine sites to date. We discovered and validated that hyper-reactive lysine residues K382 on tyrosine kinase-like orphan receptor 2 (ROR2) and K285 on Endoglin (ENG/CD105) are at the protein interaction interface in co-crystal structures of protein complexes, emphasizing the broad potential functional consequences of cell surface lysines and GASF strategy is highly desirable for discovering new active and ligandable sites that can be functionally interrogated for drug discovery.

Measuring bioavailability, utilization, and excretion of rumen-protected lysine in lactating cows using an isotope technique.

Supplementing a diet with rumen-protected amino acids (AAs) is a common feeding strategy for efficient production. For a cost-effective use of rumen-protected AA, the accurate bioavailability of rumen-protected amino acids should be known and their metabolism after absorption needs to be well understood. The current study determined the bioavailability, absorption, utilization, and excretion of rumen-protected Lys (RP-Lys). Four ruminally cannulated cows in a 4 × 4 Latin square design (12 d for diet adaptation; 5 or 6 d for total collections) received the following treatments: L0, a basal diet; L25, the basal diet and L-Lys infused into the abomasum to provide 25.9 g/d L-Lys; L50, the basal diet and L-Lys infused into the abomasum to provide 51.8 g/d L-Lys; and RPL, the basal diet supplemented with 105 g/d (as-is) of RP-Lys to provide 26.7 g of digestible Lys. During the last 5 or 6 d in each period, 15N-Lys (0.38 g/d) was infused into the abomasum for all cows to label the pool of AA, and the total collection of milk, urine, and feces were conducted. 15N enrichment of samples on d 4 and 5 were used to calculate the bioavailability and Lys metabolism. We used a model containing a fast AA turnover (≤ 5 d) and slow AA turnover pool (> 5 d) to calculate fluxes of Lys. The Lys flux to the fast AA turnover pool (absorbed Lys + Lys from the slow AA turnover pool to fast AA turnover pool) was calculated using 15N enrichment of milk Lys. The flux of Lys from the fast AA turnover pool to milk and urine was calculated using 15N transfer into milk and urine. Then, absorbed Lys was estimated by the sum of Lys flux to milk and urine assuming no net utilization of Lys by body tissues. Duodenal Lys flow was estimated by 15N enrichment of fecal Lys. The bioavailability of RP-Lys was calculated from duodenal Lys flows and Lys absorption for RPL. Increasing Lys supply from L25 to L50 increased Lys utilization for milk by 9 g/d but also increased urinary excretion by 10 g/d. For RPL, absorbed Lys was estimated to be 136 g/d where 28 g of absorbed Lys originated from RP-Lys. In conclusion, 68% of bioavailability was obtained for RP-Lys. The Lys provided from RP-Lys was not only utilized for milk protein (48%) but also excreted in urine (20%) after oxidation.

Standardized ileal digestibility of amino acids in Chinese fermented soybean meal from different sources fed to mid and late-gestating sows.

We determined apparent ileal digestibility (AID) and standardized ileal digestibility (SID) values of crude protein (CP) and amino acids (AA) in fermented soybean meal from five different sources (FSBM 1 to 5) in China when fed to mid and late-gestating sows. Twenty-four parity four sows (12 at 30 d in gestation and 12 at 80 d in gestation) were fitted with a T-cannula in the distal ileum and used in this experiment. Sows were randomly assigned to a replicated 6 × 3 Youden square design including six diets and three periods. Six diets were provided for sows in mid and late gestation, including a nitrogen-free diet and five test diets containing 26% FSBM from different sources. Results showed that there were differences in AID and SID of CP among the different FSBM samples, but no differences between sow physiological stages were observed. Specifically, when mid-gestating sows were fed FSBM 2, the AID of CP was the lowest, whereas FSBM 3 exhibited a greater AID of CP when compared to the other FSBM samples (P < 0.01). Furthermore, during late gestation, FSBM 3 consistently had greater SID of CP when compared to other FSBM samples (P < 0.01). The ileal digestibility of most AA varied with different FSBM samples. In both mid and late gestation, differences (P < 0.05) were observed for AID of lysine, tryptophan, histidine, and arginine across different FSBM samples. Similarly, the AID of dispensable AA (cysteine, glutamine, and serine) also exhibited differences (P < 0.05) across different FSBM samples in both mid and late-gestating sows. For mid-gestating sows, SID differences relating to lysine, phenylalanine, tryptophan, threonine, and arginine were observed among different diets (P < 0.05). In late-gestating sows, SID values for lysine, tryptophan, leucine, and arginine differed across diets (P < 0.05). Furthermore, the ileal digestibility of some dispensable AA was influenced by physiological stage, as evidenced by greater AID and SID values for glycine, glutamine, cysteine, and serine in late-gestating sows when compared to mid-gestating sows (P < 0.01). In summary, our study determined AA ileal digestibility of different FSBM fed to mid and late-gestating sows. We observed that the AA ileal digestibility differed among five FSBM samples, but the physiological stage of sows did not affect the ileal digestibility of CP and most AA. Additionally, when formulating diets for sows, it is crucial to consider the nutritional value differences of FSBM.

Fermented soybean meal (FSBM) is obtained from the microbial fermentation of soybean meal, which reduces anti-nutritional factor levels and enhances other nutrient content. Substituting soybean meal with FSBM in piglet and growing pig diets improves nutrient digestibility. However, its nutritional value for sows remains unclear. Therefore, five sources of FSBM were fed to sows in mid and late gestation to evaluate apparent ileal digestibility (AID) and standardized ileal digestibility (SID) values of amino acids (AA). We found that different FSBM samples impacted the SID value of AA when fed to gestating sows. Additionally, sow physiological stage influenced the SID of some dispensable AA. These findings provide valuable insights into the incorporation of FSBM into sow diets.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy. METHODS: The expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA. RESULTS: We identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth. CONCLUSIONS: This study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.

Impact of brining and drying processes on the nutritive value of tambaqui fish (Colossoma macropomum).

Preservation of fish as diet ingredient is challenging in many tropical regions due to poor socioeconomic conditions and lack of freezing facilities. So, alternative preservation techniques could be viable to address the issue. The present study evaluated the effect of brine salting (15% w/v) prior to drying at different temperatures on the nutrient profiles of tambaqui fish (Colossoma macropomum). Whole fish samples (n = 48; 792 ± 16 g; 8 months old) were grouped into two as brine-salted and non-salted, and treated at seven different drying temperatures of 30, 35, 40, 45, 50, 55 and 60°C for a period of 23 h each. To evaluate the impact of Maillard reaction, reactive lysine was also quantified. Drying temperature had no effect on the evaluated macro- and micro-nutrients of tambaqui fish (P > 0.05) while brining reduced the overall protein concentration by 6% (58.8 to 55.4 g/100 g DM; P = 0.004). Brining significantly reduced many amino acids: taurine by 56% (7.1 to 3.1 g/kg; P < 0.001), methionine 17% (14.7 to 12.1 g/kg; P < 0.001), cysteine 11% (5.1 to 4.4 g/kg, P = 0.016), and reactive lysine 11% (52.0 to 46.4 g/kg; P = 0.004). However, alanine, arginine, and serine were not affected by brining (P > 0.05). Brining also reduced the concentrations of Se by 14% (149 to 128 µg/kg DM; P = 0.020), iodine 38% (604 to 373 µg/kg DM; P = 0.020), K 42% (9.71 to 5.61 g/kg DM; P < 0.001) and Mg 18% (1.32 to 1.10 g/kg DM; P = < 0.001) versus an anticipated vast increase in Na by 744% (2.70 to 22.90 g/kg DM; P < 0.001) and ash 28% (12.4 to 16.0 g/100g DM; P < 0.001) concentration. Neither brining nor drying temperature induced changes in % lysine reactivity and fat content of tambaqui fish (P > 0.05). Agreeably, results of multivariate analysis showed a negative association between brining, Na, and ash on one side of the component and most other nutrients on the other component. In conclusion, drying without brining may better preserve the nutritive value of tambaqui fish. However, as a practical remark to the industry sector, it is recommended that the final product may further evaluated for any pathogen of economic or public health importance.

Transcriptional Expression of Histone Acetyltransferases and Deacetylases During the Recovery of Acute Exercise in Mouse Hippocampus.

Protein acetylation, which is dynamically maintained by histone acetyltransferases (HATs) and deacetylases (HDACs), might play essential roles in hippocampal exercise physiology. However, whether HATs/HDACs are imbalanced during the recovery phase following acute exercise has not been determined. Groups of exercised mice with different recovery periods after acute exercise (0 h, 0.5 h, 1 h, 4 h, 7 h, and 24 h) were constructed, and a group of sham-exercised mice was used as the control. The mRNA levels of HATs and HDACs were detected via real-time quantitative polymerase chain reaction. Lysine acetylation on the total proteins and some specific locations on histones were detected via western blotting, as were various acylation modifications on the total proteins. Except for four unaffected genes (Hdac4, Ncoa1, Ncoa2, and Sirt1), the mRNA expression trajectories of 21 other HATs or HDACs affected by exercise could be categorized into three clusters. The genes in Cluster 1 increased quickly following exercise, with a peak at 0.5 h and/or 1 h, and remained at high levels until 24 h. Cluster 2 genes presented a gradual increase with a delayed peak at 4 h or 7 h postexercise before returning to baseline. The expression of Cluster 3 genes decreased at 0.5 h and/or 1 h, with some returning to overexpression (Hdac1 and Sirt3). Although most HATs were upregulated and half of the affected HDACs were downregulated at 0.5 h postexercise, the global or residue-specific histone acetylation levels were unchanged. In contrast, the levels of several metabolism-related acylation products of total proteins, including acetylation, succinylation, 2-hydroxyisobutyryllysine, ß-hydroxybutyryllysine, and lactylation, decreased and mainly occurred on nonhistones immediately after exercise. During the 24-h recovery phase after acute exercise, the transcriptional trajectory of HATs or the same class of HDACs in the hippocampus exhibited heterogeneity. Although acute exercise did not affect the selected sites on histone lysine residues, it possibly incurred changes in acetylation and other acylation on nonhistone proteins.

EZH2 as a major histone methyltransferase in PDGF-BB-activated orbital fibroblast in the pathogenesis of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is an extra-thyroidal complication of Graves' disease which can lead to vision loss in severe cases. Currently, treatments of GO are not sufficiently effective, so novel therapeutic strategies are needed. As platelet-derived growth factor (PDGF)-BB induces several effector mechanisms in GO orbital fibroblasts including cytokine production and myofibroblast activation, this study aims to investigate the roles of histone lysine methyltransferases (HKMTs) in PDGF-BB-activated GO orbital fibroblasts by screening with HKMTs inhibitors library. From the total of twelve selective HKMT inhibitors in the library, EZH2, G9a and DOT1L inhibitors, DZNeP, BIX01294 and Pinometostat, respectively, prevented PDGF-BB-induced proliferation and hyaluronan production by GO orbital fibroblasts. However, only EZH2 inhibitor, DZNeP, significantly blocked pro-inflammatory cytokine production. For the HKMTs expression in GO orbital fibroblasts, PDGF-BB significantly and time-dependently induced EZH2, G9a and DOT1L mRNA expression. To confirm the role of EZH2 in PDGF-BB-induced orbital fibroblast activation, EZH2 silencing experiments revealed suppression of PDGF-BB-induced collagen type I and α-SMA expression along with decreasing histone H3 lysine 27 trimethylation (H3K27me3) level. In a more clinically relevant model than orbital fibroblast culture experiments, DZNeP treated GO orbital tissues significantly reduced pro-inflammatory cytokine production while slightly reduced ACTA2 mRNA expression. Our data is the first to demonstrate that among all HKMTs EZH2 dominantly involved in the expression of myofibroblast markers in PDGF-BB-activated orbital fibroblast from GO presumably via H3K27me3. Thus, EZH2 may represent a novel therapeutics target for GO.

Electrospun L-Lysine/Amorphous Calcium Phosphate Loaded Core-Sheath Nanofibers for Managing Oral Biofilm Infections and Promoting Periodontal Tissue Repairment.

Introduction: Periodontitis, a chronic inflammatory disease prevalent worldwide, is primarily treated through GTR for tissue regeneration. The efficacy of GTR, however, remains uncertain due to potential infections and the intricate microenvironment of periodontal tissue. Herein, We developed a novel core-shell structure multifunctional membrane using a dual-drug-loaded coaxial electrospinning technique (Lys/ACP-CNF), contains L-lysine in the outer layer to aid in controlling biofilms after GTR regenerative surgery, and ACP in the inner layer to enhance osteogenic performance for accelerating alveolar bone repair. Methods: The biocompatibility and cell adhesion were evaluated through CCK-8 and fluorescence imaging, respectively. The antibacterial activity was assessed using a plate counting assay. ALP, ARS, and RT-qPCR were used to examine osteogenic differentiation. Additionally, an in vivo experiment was conducted on a rat model with acute periodontal defect and infection. Micro-CT and histological analysis were utilized to analyze the in vivo alveolar bone regeneration. Results: Structural and physicochemical characterization confirmed the successful construction of the core-shell fibrous structure. Additionally, the Lys/ACP-CNF showed strong antibacterial coaggregation effects and induced osteogenic differentiation of PDLSCs in vitro. The in vivo experiment confirmed that Lys/ACP-CNF promotes new bone formation. Conclusion: Lys/ACP-CNF rapidly exhibited excellent antibacterial activity, protected PDLSCs from infection, and was conducive to osteogenesis, demonstrating its potential application for clinical periodontal GTR surgery.

Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Immunohistochemical expression of histone modification pattern in adult glioblastoma.

BACKGROUND: Despite the growing advances in molecular research and therapeutics, glioblastomas are still considered highly invasive aggressive tumors with a median survival of 15 months. Genetic alterations have been studied in detail; however, additionally, there is now growing evidence on the role of epigenetic alterations in glioblastoma. Recently, histone modification patterns have been found to have a significant part in gene expression and prognosis. However, further research in this field is warranted to establish its role for the betterment of these patients with the deadly disease. AIMS: To determine the immunohistochemical expression of histone modifications like histone-3-lysine-18 acetylation (H3K18Ac) and histone-4-lysine 20 trimethylation (H4K20triMe) in glioblastoma patients. MATERIALS AND METHODS: This is a retrospective study of 48 glioblastoma patients who underwent surgery. Immunohistochemistry (IHC) for tri-methyl-histone-H4 (Lys20) (H4K20triMe) and acetyl-histone-H3 (Lys18) (H3K18Ac) was performed in paraffin-embedded tissues manually, and the expression was noted. Data on the mitotic index and overall survival was collected and statistically analyzed. RESULTS: The mean age was 50 years with a M: F ratio of 1.6:1. Out of 48 cases, 60% (28 cases) demonstrated positivity for H3K18Ac and 98% (46 cases) for H4K20triMe. The pattern of expression was nuclear with increased expression adjacent to necrosis and at the invasive front. The overall median Q score for H3K18Ac was 1/12 and for H4K20triMe was 6/12. No significant statistical significance was observed between histone expression, Ki67%, and overall survival. CONCLUSION: Histone modification patterns are being explored in detail in an array of tumors. They also have a potential role in glioblastoma for risk stratification and instituting appropriate treatment based on the prognosis. Epigenetic changes like histone modification patterns, in addition to genetics, can pave the way for a better molecular understanding of glioblastomas and provide hope in the future to improve the survival of these patients with deadly diseases.

Global proteomic analysis reveals lysine succinylation is involved in the pathogenesis of hypertrophic scar.

Lysine succinylation (Ksucc) is a recently identified posttranslational modification that is involved in many diseases. This study examined the role of Ksucc in the pathogenesis of hypertrophic scar (HS). The presence of Ksucc in human skin was measured by immunoblotting. Ksucc occurs in many skin proteins ranging from 25 to 250 kDa, and higher levels of Ksucc are found in HS skin than in normal skin. An immunoaffinity approach coupled with LC-MS/MS was used to characterize the first succinylome of human skin, and 159 Ksucc sites in 79 proteins were identified. Among these, there were 38 increased succinylated sites in 29 proteins but no decreased succinylated sites in HS compared with normal skin. A parallel reaction monitoring assay was performed to validate the results of the succinylome and showed that the levels of Ksucc in decorin and collagens, which are involved in the pathogenesis of HS, were increased in HS than in normal skin. In addition, increasing the level of Ksucc enhanced cell proliferation and upregulated the expression of fibrosis markers (α-SMA, COL1, and COL3) in human skin fibroblasts. Our results provide global insights into the functional role of Ksucc in hypertrophic scarring.

Histone Lysine Demethylase KDM5 Inhibitor CPI-455 Induces Astrocytogenesis in Neural Stem Cells.

Lysine-specific histone demethylase 5A (KDM5A) is known to facilitate proliferation in cancer cells and maintain stemness to repress the astrocytic differentiation of neural stem cells (NSCs). In the study presented here, we investigated the effect of a KDM5 inhibitor, CPI-455, on NSC fate control. CPI-455 induced astrocytogenesis in NSCs during differentiation. Kdm5a, but not Kdm5c, knockdown induced glial fibrillary acidic protein (Gfap) transcription. CPI-455 induced signal transducer and activator of transcription 3, increased bone morphogenetic protein 2 expression, and enhanced mothers against decapentaplegic homolog 1/5/9 phosphorylation. The treatment of CPI-455 enhanced the methylation of histone H3 lysine 4 in the Gfap promoter when compared to that of the dimethyl sulfoxide control. In addition, CPI-455 treatment significantly reduced the recruitment of KDM5A to the Gfap promoter. Our data suggest that the KDM5 inhibitor CPI-455 effectively controls NSC cell fate via KDM5A inhibition and induces astrocytogenesis.

Effects of dietary protein level on intestinal function and inflammation in nursery pigs.

High crude protein (CP; 21% to 26%) diets fed during the first 21 to 28 d postweaning are viewed negatively because of a perceived increase in the incidence rates of diarrhea due to increased intestinal protein fermentation and/or augmented enteric pathogen burden. This is thought to antagonize nursery pig health and growth performance. Therefore, our objective was to evaluate the impact of low vs. high dietary CP on 21-day postweaned pig intestinal function. Analyzed parameters included ex vivo intestinal barrier integrity (ileum and colon), ileal nutrient transport, tissue inflammation, and fecal DM. One hundred and twenty gilts and barrows (average body weight) were randomly assigned to one of two diets postweaning. Diets were fed for 21 d, in two phases. Phase 1 diets: low CP (17%) with a 1.4% standardized ileal digestible (SID) Lys (LCP), or high CP (24%) with a 1.4% SID Lysine (HCP). Phase 2: LCP (17%) and a 1.35% SID lysine, or HCP (24%) formulated to a 1.35% SID lysine. Pig growth rates, feed intakes, and fecal consistency did not differ (P > 0.05) due to dietary treatment. Six animals per treatment were euthanized for additional analyses. There were no differences in colonic epithelial barrier function as measured by transepithelial electrical resistance (TER) and fluorescein isothiocyanate (FITC)-dextran transport between treatments (P > 0.05). Interleukins (IL)-1α, IL-1ß, IL-1ra, IL-2 IL-4, IL-6, and IL-12 were not different between treatments (P > 0.05). However, IL-8 and IL-18 were higher in HCP- vs. LCP-fed pigs (P < 0.05). There were no differences in fecal dry matter (DM; P > 0.05) between treatments. In the ileum, there was a tendency (P = 0.06) for TER to be higher in HCP-fed pigs, suggesting a more robust barrier. Interestingly, glucose and glutamine transport were decreased in HCP- vs. LCP-fed pigs (P < 0.05). FITC-dextran transport was not different between treatments (P > 0.05). There were also no differences in ileal cytokine concentrations between diets (P > 0.05). Taken together, the data show that low CP does not negatively impact colonic barrier function, fecal DM, or inflammation. In contrast, ileal barrier function and nutrient transport were altered, suggesting a regional effect of diet on overall intestinal function.

High dietary crude protein (CP) is thought to antagonize nursery pig enteric health. Feeding high CP diets to nursery pigs did not exacerbate intestinal health or inflammation, and overall, protein level in the diet has little impact on animal health and performance.

Lysine methyltransferase 5C increases the proliferation and metastatic abilities of clear cell renal cell carcinoma via aerobic glycolysis.

Among all types of renal cancer, clear cell renal cell carcinoma (ccRCC) is the most common and lethal subtype and is associated with a high risk of metastasis and recurrence. Histone modifications regulate several biological processes that are fundamental to the development of cancer. Lysine methyltransferase 5C (KMT5C; also known as SUV420H2) is an epigenetic modifier responsible for the trimethylation of H4K20, which drives critical cellular events, including genome integrity, cell growth and epithelial­mesenchymal transition (EMT), in various types of cancer. However, the role of KMT5C in ccRCC remains unclear. As such, the expression and function of KMT5C in ccRCC were investigated in the present study. KMT5C expression was significantly increased in ccRCC tissues compared with normal tissues (P<0.0001), and it was closely associated with the overall survival rate of patients with ccRCC. By establishing ccRCC cell lines with KMT5C expression knockdown, the role of KMT5C in the maintenance of aerobic glycolysis in ccRCC cells via the regulation of several vital glycolytic genes was identified. Additionally, KMT5C promoted the proliferation and EMT of ccRCC cells by controlling crucial EMT transcriptional factors. Together, these data suggested that KMT5C may act as an oncoprotein, guide molecular diagnosis, and shed light on novel drug development and therapeutic strategies for patients with ccRCC.

Combining directed evolution with high cell permeability for high-level cadaverine production in engineered Escherichia coli.

The biosynthesis of cadaverine from lysine is an environmentally promising technology, that could contribute to a more sustainable approach to manufacturing bio-nylon 5X. However, the titer of biosynthesized cadaverine has still not reached a sufficient level for industrial production. A powerful green cell factory was developed to enhance cadaverine production by regulating lipopolysaccharide (LPS) genes and improving membrane permeability. Firstly, 10 LPS mutant strains were constructed and the effect on the growth was investigated. Then, the lysine decarboxylase (CadA) was overexpressed in 10 LPS mutant strains of Escherichia coli MG1655 and the ability to produce cadaverine was compared. Using 20.0 g L-1 of L-lysine hydrochloride (L-lysine-HCl) as the substrate for the biotransformation reaction, Cad02 and Cad06 strains exhibited high production levels of cadaverine, with 8.95 g L-1 and 7.55 g L-1 respectively while the control strain Cad00 only 4.92 g L-1 . Directed evolution of CadA was also used to improve its stability under alkaline conditions. The cadaverine production of the Cad02-M mutant stain increased by 1.86 times at pH 8.0. Finally, the production process was scaled up using recombinant whole cells as catalysts, achieving a high titer of 211 g L-1 cadaverine (96.8%) by fed-batch bioconversion. This study demonstrates the potential role of LPS in enhancing the efficiency of mass transfer between substrate and enzymes in vivo by increasing cell permeability. The results indicate that the argumentation of cell permeability could not only significantly enhance the biotransformation efficiency of cadaverine, but also provide a universally applicable, straightforward, environment-friendly, and cost-effective method for the biosynthesis of other high-value chemicals.

Isothiocyanates Potentiate Tazemetostat-Induced Apoptosis by Modulating the Expression of Apoptotic Genes, Members of Polycomb Repressive Complex 2, and Levels of Tri-Methylating Lysine 27 at Histone 3 in Human Malignant Melanoma Cells.

In this study, we utilized an in vitro model consisting of human malignant melanoma as well as non-tumorigenic immortalized keratinocyte cells with the aim of characterizing the therapeutic effectiveness of the clinical epigenetic drug Tazemetostat alone or in combination with various isothiocyanates. In doing so, we assessed markers of cell viability, apoptotic induction, and expression levels of key proteins capable of mediating the therapeutic response. Our data indicated, for the first time, that Tazemetostat caused a significant decrease in viability levels of malignant melanoma cells in a dose- and time-dependent manner via the induction of apoptosis, while non-malignant keratinocytes were more resistant. Moreover, combinatorial treatment protocols caused a further decrease in cell viability, together with higher apoptotic rates. In addition, a significant reduction in the Polycomb Repressive Complex 2 (PRC2) members [e.g., Enhancer of Zeste Homologue 2 (EZH2), Embryonic Ectoderm Development (EED), and suppressor of zeste 12 (SUZ12)] and tri-methylating lysine 27 at Histone 3 (H3K27me3) protein expression levels was observed, at least partially, under specific combinatorial exposure conditions. Reactivation of major apoptotic gene targets was determined at much higher levels in combinatorial treatment protocols than Tazemetostat alone, known to be involved in the induction of intrinsic and extrinsic apoptosis. Overall, we developed an optimized experimental therapeutic platform aiming to ensure the therapeutic effectiveness of Tazemetostat in malignant melanoma while at the same time minimizing toxicity against neighboring non-tumorigenic keratinocyte cells.

Proanthocyanidins Ameliorate LPS-Inhibited Osteogenesis of PDLSCs by Restoring Lysine Lactylation.

Periodontitis is a bacteria-induced inflammatory disease characterized by the progressive destruction of periodontal supporting tissues. Periodontal ligament stem cells (PDLSCs) are capable of differentiating into osteoblasts, which is an important stem cell source for endogenous periodontal tissue regeneration. Lysine lactylation (Kla) is a novel post-translational modification of proteins that is recently thought to be associated with osteogenic differentiation. Here, we found that lactylation levels are reduced both in the periodontal tissue of rats with periodontitis and lipopolysaccharide (LPS)-stimulated human PDLSCs. Proanthocyanidins were able to promote the osteogenesis of inflamed PDLSCs by restoring lactylation levels. Mechanistically, proanthocyanidins increased lactate production and restored the lactylation levels of PDLSCs, which recovered osteogenesis of inflamed PDLSCs via the Wnt/ß-catenin pathway. These results provide evidence on how epigenetic regulation by pharmacological agents influence the osteogenic phenotype of stem cells and the process of periodontal tissue repair. Our current study highlights the valuable potential of natural product proanthocyanidins in the regenerative engineering of periodontal tissues.

Lysine demethylase LSD1 is associated with stemness in EBV-positive B cell lymphoma.

EBV-infected lymphoma has a poor prognosis and various treatment strategies are being explored. Reports suggesting that B cell lymphoma can be induced by epigenetic regulation have piqued interest in studying mechanisms targeting epigenetic regulation. Here, we set out to identify an epigenetic regulator drug that acts synergistically with doxorubicin in EBV-positive lymphoma. We expressed the major EBV protein, LMP1, in B-cell lymphoma cell lines and used them to screen 100 epigenetic modifiers in combination with doxorubicin. The screening results identified TCP, which is an inhibitor of LSD1. Further analyses revealed that LMP1 increased the activity of LSD1 to enhance stemness ability under doxorubicin treatment, as evidenced by colony-forming and ALDEFLUOR activity assays. Quantseq 3' mRNA sequencing analysis of potential targets regulated by LSD1 in modulating stemness revealed that the LMP1-induced upregulation of CHAC2 was decreased when LSD1 was inhibited by TCP or downregulated by siRNA. We further observed that SOX2 expression was altered in response to CHAC2 expression, suggesting that stemness is regulated. Collectively, these findings suggest that LSD1 inhibitors could serve as promising therapeutic candidates for EBV-positive lymphoma, potentially reducing stemness activity when combined with conventional drugs to offer an effective treatment approach.