Search Results (17)

Phosphoethanolamine N-methyltransferase (PEAMT) is a key enzyme that catalyzes three successive methylation steps of phosphoethanolamine (P-EA) to phosphocholine (P-Cho). Meanwhile, P-Cho is a major precursor of phosphatidylcholine (PC) and glycine betaine (GB), which are involved in cell signal transduction, stress response, etc. Therefore, the PEAMT gene plays an essential role in plant growth and development as well as stress resistance. There are two homologous PEAMT genes in rice (Oryza sativa L. ssp. japonica), namely, OsPEAMT1 and OsPEAMT2. However, there has not been any comprehensive functional analysis of these two genes. Here, we employed bioinformatics methods to analyze the amino acid sequences and promoters of OsPEAMT1 and OsPEAMT2, and found that both proteins contain two methyltransferase domains. OsPEAMT1 is highly similar with ZmPEAMT, and OsPEAMT2 is closely related to LmPEAMT and TaPEAMT. There are abundant plant hormone response elements, stress response elements and low-temperature response elements in the promoters of OsPEAMT1 and OsPEAMT2. The in vitro enzymatic activity assays of recombinant proteins of OsPEAMT1 and OsPEAMT2 indicated that they can catalyze the production of P-Cho from P-EA, respectively. Meanwhile, the endogenous P-Cho content increased significantly (p < 0.05) when exogenous P-EA was added to rice. These indicate that OsPEAMT1 and OsPEAMT2 proteins have catalytic functions in vivo and in vitro. The expression patterns of both genes are different in different tissues, flowers and seeds at various developmental stages. Additionally, both genes have different responses to salt and low-temperature stress. This study supplies valuable insights into the function of OsPEAMT1 and OsPEAMT2, and it will provide key targets for rice molecular breeding, offering important insights for the development of rice with stress resistance and high yield. Full article

The Muong La–Bac Yen–Cho Bo fault is one of the seismic faults in the northwest region of Vietnam. Neo-tectonic activities and exogenous processes have influenced the drainage system and topographic–geomorphologic features on both walls of the fault. The results of topographic analysis and geomorphological indices have confirmed the active tectonics of the fault during the Neo-tectonic period (Pliocene–Quaternary, about 5 million years). The valley floor width-to-height ratio (V_f) of less than 0.5 indicates the “rejuvenation” of the streams and the obvious influence of tectonic activities on the two walls of the studied fault. The Hypsometric curve (HC) in the study area has a straight–convex shape and the basins on the northeast wall have hypsometric integral index values ranging from 0.46 to 0.481, which are lower and more convex than those of the southwest wall. The Mountain-front sinuosity index (S_mf) from 1.92 to 3.28 along the foot slope of the Hoang Lien Son range (the segment from Than Uyen to Bac Yen and Phu Yen) and the highly variable stream-length gradient index (SL) value on the northeast wall signify the relative tectonic uplift on the northeast wall of the fault. The deformed geomorphological indications (steep cliffs, slip surfaces, etc.) in the field confirm the active tectonics of the Muong La–Bac Yen–Cho Bo fault during the Neo-tectonic period. Full article

Objectives: This article compares metabolic, pancreatic, and gut-derived hormone responses to isomaltulose ingestion, before versus during submaximal sustained exercise, in adults with type 1 diabetes (T1D) using automated insulin delivery systems. Methods: In a randomized, cross-over trial, eight participants with T1D being treated with automated insulin pumps (five females, age: 47 ± 16 years, BMI: 27.5 ± 3.8 kg·m², diabetes duration: 23 ± 11 years, HbA1c: 8.3 ± 0.9 [67.5 ± 9.5]% [mmol/mol]) attended the laboratory on two separate occasions and consumed an isocaloric amount of isomaltulose as either (1) a single serving (0.75g CHO·kg⁻¹ BM) with a 25% reduction in bolus insulin 90 min before 45 min of cycling (PEC) or (2) three separate isocaloric servings (0.25g CHO·kg⁻¹ BM each) without bolus insulin during exercise (DEC). Plasma glucose (PG), gut incretins (GLP-1 and GIP), pancreatic glucagon, exogenous insulin, and whole-body fuel oxidation rates were determined. Data were treated via a two-way repeated measures ANOVA, with p ≤ 0.05 accepted as significant. Results: PG concentrations throughout exercise were higher and less variable with DEC compared to PEC. The exercise-induced change in PG was directionally divergent between trials (PEC: ∆ − 3.2 ± 1.2 mmol/L vs. DEC: ∆ + 1.7 ± 1.5 mmol/L, p < 0.001), changing at a rate of −0.07 ± 0.03 mmol/L/min with PEC and +0.04 ± 0.03 mmol/L/min with DEC (p < 0.001 between conditions). Throughout the exercise period, GLP-1, GIP, glucagon, and total insulin concentrations were lower with DEC (all p ≤ 0.02). The oxidation rates of carbohydrates were lower (p = 0.009) and of lipids were greater (p = 0.014) with DEC compared to PEC. Conclusions: The consumption of smaller servings of isomaltulose during, rather than as a single isocaloric serving before, submaximal sustained exercise provided (i) a better glycemic protective effect, (ii) a lesser push on pancreatic and gut-mediated glucoregulatory hormones, and (iii) a lower reliance on whole-body carbohydrate oxidation. Such information serves to remind us of the potential importance of nutrition for modulating the metabolic fate of an acute bout of exercise and may help inform best practice guidelines for exercise management in the T1D-sphere. Full article

The phenolic aldehyde syringaldehyde (SA) has been shown to have an antihyperglycemic effect in diabetic rats due to increased glucose utilization and insulin sensitivity. To understand the direct effect of SA on the GLP-1 receptor, STZ-induced diabetic rats were used. The levels of pro-inflammatory cytokines, liver enzymes, and renal function were measured using specific ELISA kits. The mechanisms of SA effects were investigated using CHO-K1 cells, pancreatic Min-6 cells, and cardiomyocyte H9c2 cells. The results indicated that the antihyperglycemic effect of SA in diabetic rats was abolished by blocking the GLP-1 receptor with an antagonist. SA has a direct effect on the GLP-1 receptor when using CHO-K1 cells transfected with the exogenous GLP-1 receptor gene. In addition, SA stimulated insulin production in Min-6 cells by activating GLP-1 receptors. SA caused a dose-dependent rise in GLP-1 receptor mRNA levels in cardiac H9c2 cells. These in vitro results support the notion that SA has a direct effect on the GLP-1 receptor. Otherwise, SA inhibited the increase of pro-inflammatory cytokines, including interleukins and tumor TNF-α, in type 1 diabetic rats in a dose-dependent manner. Moreover, as with liraglutide, SA reduced plasma lipid profiles, including total cholesterol and triglyceride, in mixed diet-induced type 2 diabetic rats. Intriguingly, chronic treatment with SA (as with liraglutide) reversed the functions of both the liver and the kidney in these diabetic rats. SA displayed less efficiency in reducing body weight and food consumption compared to liraglutide. In conclusion, SA effectively activates GLP-1 receptors, resulting in a reduction in diabetic-related complications in rats. Therefore, it is beneficial to develop SA as a chemical agonist for clinical applications in the future. Full article

Relative to carbohydrate (CHO) alone, exogenous ketones followed by CHO supplementation during recovery from glycogen-lowering exercise have been shown to increase muscle glycogen resynthesis. However, whether this strategy improves subsequent exercise performance is unknown. The objective of this study was to assess the efficacy of ketone monoester (KME) followed by CHO ingestion after glycogen-lowering exercise on subsequent 20 km (TT_20km) and 5 km (TT_5km) best-effort time trials. Nine recreationally active men (175.6 ± 5.3 cm, 72.9 ± 7.7 kg, 28 ± 5 y, 12.2 ± 3.2% body fat, VO₂max = 56.2 ± 5.8 mL· kg BM⁻¹·min⁻¹; mean ± SD) completed a glycogen-lowering exercise session, followed by 4 h of recovery and subsequent TT_20km and TT_5km. During the first 2 h of recovery, participants ingested either KME (25 g) followed by CHO at a rate of 1.2 g·kg⁻¹·h⁻¹ (KME + CHO) or an iso-energetic placebo (dextrose) followed by CHO (PLAC + CHO). Blood metabolites during recovery and performance during the subsequent two-time trials were measured. In comparison to PLAC + CHO, KME + CHO displayed greater (p < 0.05) blood beta-hydroxybutyrate concentration during the first 2 h, lower (p < 0.05) blood glucose concentrations at 30 and 60 min, as well as greater (p < 0.05) blood insulin concentration 2 h following ingestion. However, no treatment differences (p > 0.05) in power output nor time to complete either time trial were observed vs. PLAC + CHO. These data indicate that the metabolic changes induced by KME + CHO ingestion following glycogen-lowering exercise are insufficient to enhance subsequent endurance time trial performance. Full article

Chinese hamster ovary (CHO) cells are a popular choice in biopharmaceuticals because of their beneficial traits, including high-density suspension culture, safety, and exogenously produced proteins that closely resemble natural proteins. Nevertheless, a decline in the expression of exogenous proteins is noted as culture time progresses. This is a consequence of foreign gene recombination into chromosomes by random integration. The current investigation employs CRISPR-Cas9 technology to integrate foreign genes into a particular chromosomal location for sustained expression. Results demonstrate the successful integration of enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) near base 434814407 on chromosome NC_048595.1 of CHO-K1 cells. Over 60 successive passages, monoclonal cell lines were produced that consistently expressed all relevant external proteins without discernible variation in expression levels. In conclusion, the CHO-K1 cell locus, NC_048595.1, proves an advantageous locus for stable exogenous protein expression. This study provides a viable approach to establishing a CHO cell line capable of enduring reliable exogenous protein expression. Full article

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeted monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C₄₄Mab-94; IgG₁, kappa) recognized the variant-8-encoded region and peptide, indicating that C₄₄Mab-94 is a specific mAb for CD44v8. Furthermore, C₄₄Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C₄₄Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells. These results indicate that C₄₄Mab-94 is useful for detecting CD44v8 in a variety of experimental methods and is expected to become usefully applied to GC diagnosis and therapy. Full article

The aim of this systematic review with meta-analysis was to examine the influence of exogenous factors related to nutritional and hydration strategies and environmental conditions, as modulators of fatigue, including factors associated with performance fatigability and perceived fatigability, in endurance tests lasting 45 min to 3 h. A search was carried out using four databases: PubMed, Web of Science, SPORTDiscus, and EBSCO. A total of 5103 articles were screened, with 34 included in the meta-analysis. The review was registered with PROSPERO (CRD42022327203) and adhered to the PRISMA guidelines. The study quality was evaluated according to the PEDro score and assessed using Rosenthal’s fail-safe N. Carbohydrate (CHO) intake increased the time to exhaustion (p < 0.001) and decreased the heart rate (HR) during the test (p = 0.018). Carbohydrate with protein intake (CHO + PROT) increased lactate during the test (p = 0.039). With respect to hydration, dehydrated individuals showed a higher rate of perceived exertion (RPE) (p = 0.016) and had a higher body mass loss (p = 0.018). In hot conditions, athletes showed significant increases in RPE (p < 0.001), HR (p < 0.001), and skin temperature (p = 0.002), and a decrease in the temperature gradient (p < 0.001) after the test. No differences were found when athletes were subjected to altitude or cold conditions. In conclusion, the results revealed that exogenous factors, such as nutritional and hydration strategies, as well as environmental conditions, affected fatigue in endurance sports, including factors associated with performance fatigability and perceived fatigability. Full article

Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker and plays tumor promotion and drug resistance roles in various cancers. In particular, the splicing variants are overexpressed in many carcinomas and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant’s (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C₄₄Mab-3; IgG₁, kappa) recognized peptides of the variant-5-encoded region, indicating that C₄₄Mab-3 is a specific mAb for CD44v5. Moreover, C₄₄Mab-3 reacted with CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent K_D of C₄₄Mab-3 for CHO/CD44v3–10 and PK-1 was 1.3 × 10⁻⁹ M and 2.6 × 10⁻⁹ M, respectively. C₄₄Mab-3 could detect the exogenous CD44v3–10 and endogenous CD44v5 in Western blotting and stained the formalin-fixed paraffin-embedded pancreatic cancer cells but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C₄₄Mab-3 is useful for detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic cancer diagnosis and therapy. Full article

Home-based resistance exercise (RE) has become increasingly prevalent, but its effects on protein metabolism are understudied. We tested the effect of an essential amino acid formulation (EAA+: 9 g EAAs, 3 g leucine) and branched-chain amino acids (BCAAs: 6 g BCAAs, 3 g leucine), relative to a carbohydrate (CHO) placebo, on exogenous leucine retention and myofibrillar protein breakdown following dynamic bodyweight RE in a home-based setting. Twelve recreationally active adults (nine male, three female) participated in a double-blind, placebo-controlled, crossover study with four trial conditions: (i) RE and EAA+ (EX-EAA+); (ii) RE and BCAAs (EX-BCAA); (iii) RE and CHO placebo (EX-CHO); and (iv) rest and CHO placebo (REST-CHO). Total exogenous leucine oxidation and retention (estimates of whole-body anabolism) and urinary 3-methylhistidine:creatinine ratio (3MH:Cr; estimate of muscle catabolism) were assessed over 5 h post-supplement. Total exogenous leucine oxidation and retention in EX-EAA+ and EX-BCAA did not significantly differ (p = 0.116) but were greater than EX-CHO (p < 0.01). There was a main effect of condition on urinary 3MH:Cr (p = 0.034), with post hoc analysis revealing a trend (p = 0.096) for reduced urinary 3MH:Cr with EX-EAA+ (32%) compared to EX-CHO. By direct comparison, urinary 3MH:Cr was significantly lower (23%) in EX-EAA+ than EX-BCAA (p = 0.026). In summary, the ingestion of EAA+ or BCAA provided leucine that was ~60% retained for protein synthesis following home-based bodyweight RE, but EAA+ most effectively attenuated myofibrillar protein breakdown. Full article

We implemented a multi-pronged strategy (MAX) involving chronic (2 weeks high carbohydrate [CHO] diet + gut-training) and acute (CHO loading + 90 g·h⁻¹ CHO during exercise) strategies to promote endogenous and exogenous CHO availability, compared with strategies reflecting lower ranges of current guidelines (CON) in two groups of athletes. Nineteen elite male race walkers (MAX: 9; CON:10) undertook a 26 km race-walking session before and after the respective interventions to investigate gastrointestinal function (absorption capacity), integrity (epithelial injury), and symptoms (GIS). We observed considerable individual variability in responses, resulting in a statistically significant (p < 0.001) yet likely clinically insignificant increase (Δ 736 pg·mL⁻¹) in I-FABP after exercise across all trials, with no significant differences in breath H₂ across exercise (p = 0.970). MAX was associated with increased GIS in the second half of the exercise, especially in upper GIS (p < 0.01). Eighteen highly trained male and female distance runners (MAX: 10; CON: 8) then completed a 35 km run (28 km steady-state + 7 km time-trial) supported by either a slightly modified MAX or CON strategy. Inter-individual variability was observed, without major differences in epithelial cell intestinal fatty acid binding protein (I-FABP) or GIS, due to exercise, trial, or group, despite the 3-fold increase in exercise CHO intake in MAX post-intervention. The tight-junction (claudin-3) response decreased in both groups from pre- to post-intervention. Groups achieved a similar performance improvement from pre- to post-intervention (CON = 39 s [95 CI 15–63 s]; MAX = 36 s [13–59 s]; p = 0.002). Although this suggests that further increases in CHO availability above current guidelines do not confer additional advantages, limitations in our study execution (e.g., confounding loss of BM in several individuals despite a live-in training camp environment and significant increases in aerobic capacity due to intensified training) may have masked small differences. Therefore, athletes should meet the minimum CHO guidelines for training and competition goals, noting that, with practice, increased CHO intake can be tolerated, and may contribute to performance outcomes. Full article

Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity. Full article

Given the importance of exercise economy to endurance performance, we implemented two strategies purported to reduce the oxygen cost of exercise within a 4 week training camp in 21 elite male race walkers. Fourteen athletes undertook a crossover investigation with beetroot juice (BRJ) or placebo (PLA) [2 d preload, 2 h pre-exercise + 35 min during exercise] during a 26 km race walking at speeds simulating competitive events. Separately, 19 athletes undertook a parallel group investigation of a multi-pronged strategy (MAX; n = 9) involving chronic (2 w high carbohydrate [CHO] diet + gut training) and acute (CHO loading + 90 g/h CHO during exercise) strategies to promote endogenous and exogenous CHO availability, compared with strategies reflecting lower ranges of current guidelines (CON; n = 10). There were no differences between BRJ and PLA trials for rates of CHO (p = 0.203) or fat (p = 0.818) oxidation or oxygen consumption (p = 0.090). Compared with CON, MAX was associated with higher rates of CHO oxidation during exercise, with increased exogenous CHO use (CON; peak = ~0.45 g/min; MAX: peak = ~1.45 g/min, p < 0.001). High rates of exogenous CHO use were achieved prior to gut training, without further improvement, suggesting that elite athletes already optimise intestinal CHO absorption via habitual practices. No differences in exercise economy were detected despite small differences in substrate use. Future studies should investigate the impact of these strategies on sub-elite athletes’ economy as well as the performance effects in elite groups. Full article

The tightly localized noradrenergic neurons (NA) in the locus coeruleus (LC) are well recognized as essential for focused arousal and novelty-oriented responses, while many children with autism spectrum disorder (ASD) exhibit diminished attention, engagement and orienting to exogenous stimuli. This has led to the hypothesis that atypical LC activity may be involved in ASD. Oxytocin (OXT) neurons and receptors are known to play an important role in social behavior, pair bonding and cognitive processes and are under investigation as a potential treatment for ASD. However, little is known about the neurotransmission from hypothalamic paraventricular (PVN) OXT neurons to LC NA neurons. In this study, we test, in male and female rats, whether PVN OXT neurons excite LC neurons, whether oxytocin is released and involved in this neurotransmission, and whether activation of PVN OXT neurons alters novel object recognition. Using “oxytocin sniffer cells” (CHO cells that express the human oxytocin receptor and a Ca indicator) we show that there is release of OXT from hypothalamic PVN OXT fibers in the LC. Optogenetic excitation of PVN OXT fibers excites LC NA neurons by co-release of OXT and glutamate, and this neurotransmission is greater in males than females. In male, but not in female animals, chemogenetic activation of PVN OXT neurons increases attention to novel objects. Full article

Carbohydrate (CHO) ingestion during exercise lasting less than three hours improves endurance exercise performance but there is still debate about the optimal dose. We utilised stable isotopes and blood metabolite profiles to further examine metabolic responses to CHO (glucose only) ingestion in the 20–64 g·h⁻¹ range, and to determine the association with performance outcome. In a double-blind, randomized cross-over design, male cyclists (n = 20, mean ± SD, age 34 ± 10 years, mass 75.8 ± 9 kg, peak power output 394 ± 36 W, VO_2max 62 ± 9 mL·kg⁻¹·min⁻¹) completed four main experimental trials. Each trial involved a two-hour constant load ride (185 ± 25 W) followed by a time trial, where one of three CHO beverages, or a control (water), were administered every 15 min, providing 0, 20, 39 or 64 g CHO·h⁻¹. Dual glucose tracer techniques, indirect calorimetry and blood analyses were used to determine glucose kinetics, exogenous CHO oxidation (EXO), endogenous CHO and fat oxidation; and metabolite responses. Regression analysis revealed that total exogenous CHO oxidised in the second hour of exercise, and suppression of serum NEFA concentration provided the best prediction model of performance outcome. However, the model could only explain ~19% of the variance in performance outcome. The present data demonstrate that consuming ~40 g·h⁻¹ of CHO appears to be the minimum ingestion rate required to induce metabolic effects that are sufficient to impact upon performance outcome. These data highlight a lack of performance benefit and few changes in metabolic outcomes beyond an ingestion rate of 39 g·h⁻¹. Further work is required to explore dose-response effects of CHO feeding and associations between multiple metabolic parameters and subsequent performance outcome. Full article