Search Results (84)

Background/Objectives: Blunt cardiac injury (BCI) is a severe medical condition that may arise as a result of various traumas, including motor vehicle accidents and falls. The main objective of this study was to explore the role and underlying mechanisms of the TRPV4 gene in BCI. Elucidating the function of TRPV4 in BCI may reveal potential novel therapeutic targets for the treatment of this condition. Methods: Rats in each group, including the SD control group (SDCON), the SD blunt-trauma group (SDBT), the TRPV4 gene-knockout control group (KOCON), and the TRPV4 gene-knockout blunt-trauma group (KOBT), were all freely dropped from a fixed height with a weight of 200 g and struck in the left chest with a certain energy, causing BCI. After the experiment, the levels of serum IL-6 and IL-1β were detected to evaluate the inflammatory response. The myocardial tissue structure was observed by HE staining. In addition, cardiac transcriptome analysis was conducted to identify differentially expressed genes, and metabolomics studies were carried out using UHPLC-Q-TOF/MS technology to analyze metabolites. The results of transcriptomics and metabolomics were verified by qRT-PCR and Western blot analysis. Results: Compared with the SDCON group, the levels of serum IL-6 and IL-1β in the SDBT group were significantly increased (p < 0.001), while the levels of serum IL-6 and IL-1β in the KOBT group were significantly decreased (p < 0.001), indicating that the deletion of the TRPV4 gene alleviated the inflammation induced by BCI. HE staining showed that myocardial tissue injury was severe in the SDBT group, while myocardial tissue structure abnormalities were mild in the KOBT group. Transcriptome analysis revealed that there were 1045 upregulated genes and 643 downregulated genes in the KOBT group. These genes were enriched in pathways related to inflammation, apoptosis, and tissue repair, such as p53, apoptosis, AMPK, PPAR, and other signaling pathways. Metabolomics studies have found that TRPV4 regulates nucleotide metabolism, amino-acid metabolism, biotin metabolism, arginine and proline metabolism, pentose phosphate pathway, fructose and mannose metabolism, etc., in myocardial tissue. The combined analysis of metabolic and transcriptional data reveals that tryptophan metabolism and the protein digestion and absorption pathway may be the key mechanisms. The qRT-PCR results corroborated the expression of key genes identified in the transcriptome sequencing, while Western blot analysis validated the protein expression levels of pivotal regulators within the p53 and AMPK signaling pathways. Conclusions: Overall, the deletion of the TRPV4 gene effectively alleviates cardiac injury by reducing inflammation and tissue damage. These findings suggest that TRPV4 may become a new therapeutic target for BCI, providing new insights for future therapeutic strategies. Full article

E. coli attaches to, and forms biofilms on various surfaces, including latex and polystyrene, contributing to nosocomial spread. E. coli responds to both exogenous and endogenous insulin, which induces behavioral changes. Human insulin, a quorum signal surrogate for microbial insulin, may affect the ability of E. coli to interact with latex and polystyrene in the presence of various sugars. E. coli ATCC 25923 was grown in peptone (1%) yeast nitrogen base broth to either the logarithmic or stationary growth phase. Adherence to latex was determined using 6 × 6 mm latex squares placed in a suspension of washed cells (10³ CFU/mL; 30 min; 37 °C) in buffer containing insulin at 2, 20, and 200 µU/mL (Humulin^® R; Lilly) with and without mannose, galactose, fructose, sorbose, arabinose, xylose, lactose, maltose, melibiose, glucose-6-phosphate, glucose-1-phosphate, and glucosamine at concentrations reported to affect behavioral response. Attachment levels to latex were determined by the press plate method. Biofilm levels were measured in a similar fashion but with overnight cultures in flat bottom uncoated polystyrene plates. Controls were media, insulin, sugar, or buffer alone. Glucose served as the positive control. Overall, the stationary phase cells’ adherence to latex was greater, regardless of the test condition, than was measured for the logarithmic phase cells. The effect of insulin on adherence to latex was insulin and sugar concentration dependent. The addition of insulin (200 µU/mL) resulted in a significantly (p < 0.05) increased adherence to latex and biofilm formation on polystyrene compared with sugar alone for 12 of the 13 sugars tested with stationary phase bacteria and 10 of the 13 sugars tested with logarithmic phase bacteria. Adherence in response to sorbose was the only sugar tested that was unaffected by insulin. These findings show that insulin enhances E. coli’s association with materials in common usage in medical environments in a nutrition-dependent manner. Full article

Machine learning (ML) algorithms hold the potential to outperform the selection of patients for immunotherapy (ICIs) compared to previous biomarker studies. We analyzed the predictive performance of ML models and compared them to traditional clinical biomarkers (TCBs) in the field of gastrointestinal (GI) cancers. The study has been registered in PROSPERO (number: CRD42023465917). A systematic search of PubMed was conducted to identify studies applying different ML algorithms to GI cancer patients treated with ICIs using tumor RNA gene expression profiles. The outcomes included were response to immunotherapy (ITR) or survival. Additionally, we compared the ML methodology details and predictive power inherent in the published gene sets using 5-fold cross-validation and logistic regression (LR), on an available well-defined ICI-treated metastatic gastric cancer (GC) cohort (n = 45). A set of standard clinical ICI biomarkers (MLH, MSH, and CD8 genes, plus PMS2 and PD-L1)) and de-novo calculated principal components (PCs) of the original datasets were also included as additional points of comparison. Nine articles were identified as eligible to meet the inclusion criteria. Three were pan-cancer studies, five assessed GC, and one studied colorectal cancer (CRC). Classification and regression models were used to predict ICI efficacy. Next, using LR, we validated the predictive power of applied ML algorithms on RNA signatures, using their reported receiver operating characteristics (ROC) analysis area under the curve (AUC) values on a well-defined ICI-treated gastric cancer (GC) dataset (n = 45). In two cases our method has outperformed the published results (reported/LR comparison: 0.74/0.831, 0.67/0.735). Besides the published studies, we have included two benchmarks: a set of TCBs and using principal components based on the whole dataset (PCA, 99% explained variance, 40 components). Interestingly, a study using a selected gene set (immuno-oncology panel) with AUC = 0.83 was the only one that outperformed the TCB (AUC = 0.8) and the PCA (AUC =0.81) results. Cross-validation of the predictive performance of these genes on the same GC dataset and an investigation of their prognostic role on a collated multi-cohort GC dataset of n = 375 resected, or chemotherapy-treated patients revealed that genes mannose-6-phosphate receptor (M6PR), Indoleamine 2,3-Dioxygenase 1 (IDO1), Neuropilin-1 (NRP1), and MAGEA3 performed similarly, or better than established biomarkers like PD-L1 and MSI. We found an immuno-oncology panel with an AUC = 0.83 that outperformed the clinical benchmark or the PC results. We recommend further investigation and experimental validation in the case of M6PR, IDO1, NRP1, and MAGEA3 expressions based on their strong predictive power in GC ITR. Well-designed studies with larger sample sizes and nonlinear ML models might help improve biomarker selections. Full article

Phosphomannomutase 2 (PMM2) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate, a key step in the biosynthesis of GDP-mannose for N-glycosylation. Its deficiency is the most common cause of congenital disorders of glycosylation (CDGs), accounting for the subtype known as PMM2-CDG. PMM2-CDG is a rare autosomal recessive disease characterized by multisystemic dysfunction, including cerebellar atrophy, peripheral neuropathy, developmental delay, and coagulation abnormalities. The disease is associated with a spectrum of pathogenic missense mutations, particularly at residues involved in dimerization and catalytic function (i.e., p.Phe119Leu and p.Arg141His). The dimerization of PMM2 is considered essential for enzymatic activity, although it remains unclear whether this supports structural stability alone, or whether both subunits are catalytically active—a distinction that may affect how mutations in each monomer contribute to overall enzyme function and disease phenotype. PMM2 has a paralog, phosphomannomutase 1 (PMM1), which shares substantial structural similarity—including obligate dimerization—and displays mutase activity in vitro, but does not compensate for PMM2 deficiency in vivo. To investigate potential heterodimerization between PMM1 and PMM2 and the effect of interface mutations over PMM2 dimer stability, we first assessed the likelihood of their co-expression using data from GTEx and the Human Protein Atlas. Building on this expression evidence, we modeled all possible dimeric combinations between the two paralogs using AlphaFold3. Models of the PMM2 and PMM1 homodimers were used as internal controls and aligned closely with their respective reference biological assemblies (RMSD < 1 Å). In contrast, the PMM2/PMM1 heterodimer model, the primary result of interest, showed high overall confidence (pLDDT > 90), a low inter-chain predicted alignment error (PAE∼1 Å), and robust interface confidence scores (iPTM = 0.80). Then, we applied PISA, PRODIGY, and mmCSM-PPI to assess interface energetics and evaluate the impact of missense variants specifically at the dimerization interface. Structural modeling suggested that PMM2/PMM1 heterodimers were energetically viable, although slightly less stable than PMM2 homodimers. Interface mutations were predicted to reduce dimer stability, potentially contributing to the destabilizing effects of disease-associated variants. These findings offer a structural framework for understanding PMM2 dimerization, highlighting the role of interface stability, paralogs co-expression, and sensitivity to disease-associated mutations. Full article

Mucolipidosis type II (ML II) is a rare and fatal disease of acid hydrolase trafficking. It is caused by pathogenic variants in the GNPTAB gene, leading to the absence of GlcNAc-1-phosphotransferase activity, an enzyme that catalyzes the first step in the formation of the mannose 6-phosphate (M6P) tag, essential for the trafficking of most lysosomal hydrolases. Without M6P, these do not reach the lysosome, which accumulates undegraded substrates. The lack of samples and adequate disease models limits the investigation into the pathophysiological mechanisms of the disease and potential therapies. Here, we report the generation and characterization of an ML II induced pluripotent stem cell (iPSC) line carrying the most frequent ML II pathogenic variant [NM_024312.5(GNPTAB):c.3503_3504del (p.Leu1168fs)]. Skin fibroblasts were successfully reprogrammed into iPSCs that express pluripotency markers, maintain a normal karyotype, and can differentiate into the three germ layers. Furthermore, ML II iPSCs showed a phenotype comparable to that of the somatic cells that originated them in terms of key ML II hallmarks: lower enzymatic activity of M6P-dependent hydrolases inside the cells but higher in conditioned media, and no differences in an M6P-independent hydrolase and accumulation of free cholesterol. Thus, ML II iPSCs constitute a novel model for ML II disease, with the inherent iPSC potential to become a valuable model for future studies on the pathogenic mechanisms and testing potential therapeutic approaches. Full article

Lysosomal enzymes are synthesized as N-glycosylated glycoproteins with mannose-6-phosphate (M6P) moieties, which are responsible for their binding to M6P receptors and transporting to the lysosome. In the M6P biosynthetic pathway, a Man₈GlcNAc₂ glycoform is converted to M6P groups through two consecutive enzymatic reactions, including N-acetylglucosamine (GlcNAc)-1-phosphotransferase (GNPT), transferring GlcNAc-1-phosphate from UDP-GlcNAc to the C6 hydroxyl groups of mannose residues, and then, removal of the covering GlcNAc moiety from the GlcNAc-P-mannose phosphodiester was carried out using an α-N-acetylglucosaminidase (referred to as ‘uncovering enzyme’, UCE) in the trans-Golgi network (TGN). Here, we expressed differently tailored versions of the UCE, including four truncated variants, in Escherichia coli. The four variants with the signal peptide, transmembrane domain, propiece and cytoplasmic tail truncated, respectively, were purified by affinity chromatography, and their enzymatic activities were assayed using a UDP-Glo kit. By fusing a maltose-binding protein (MBP) in the N-terminus of the UCE variants, the fusion proteins could be soluble when expressed in E. coli. The highest concentration of the purified enzyme was 80.5 mg/L of fermentation broth. Furthermore, the UCE with the core catalytic domain exhibited the highest uncovering activity. Full article

Mutations in GNPTAB underlie mucolipidosis II and mucolipidosis III α/β, which are inherited lysosomal storage disorders caused by a defective UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine phosphotransferase. As a result, newly synthesized acid hydrolases fail to acquire Mannose-6-Phosphate (Man-6-P) sorting signals, or do so to a lesser extent, and exhibit an impaired trafficking to lysosomes. Interestingly, we found that GNPTAB knockout HeLa cells are resistant to several cytotoxic agents: doxorubicin, chloroquine, staurosporine and paclitaxel. While we detected an increased trapping of weak bases in the expanded lysosomal population of these cells, which could reduce the effect of doxorubicin and chloroquine; the decreased cell response to staurosporine and paclitaxel suggested the involvement of alternative resistance mechanisms. Indeed, further investigation revealed that the hyperactivation of the Insulin-like Growth Factor 1 Receptor (IGF1R) pathway is a central player in the apoptosis resistance exhibited by Man-6-P sorting deficient cells. Full article

Mucolipidosis type II is a very rare lysosomal disease affecting the UDP-GlcNAc N-acetylglucosamine-1-phosphotransferase enzyme, which catalyzes the synthesis of the targeting signal mannose 6-phosphate in lysosomal acid hydrolases. Its deficiency hinders the arrival of lysosomal enzymes to the lysosome, diminishing the multiple degradations of components that cells need to perform. Due to the low prevalence of this condition, available information is scarce. This article aims to deepen the understanding of the disease; clinical, biochemical, and proteomic data are analyzed. Three patients have been identified presenting GNPTAB pathogenic variants using whole exome sequencing. A biochemical profile for these patients has been carried out through quantification of glycosaminoglycans in urine samples and enzymatic analysis in dried blood spot (DBS) samples. Quantitative proteomic studies were performed. Results show how enzymatic assays in DBS can be used to diagnose this disease both during the neonatal period or in patients of more advanced age. Increased levels of acid sphingomyelinase, alpha-iduronidase, iduronidate 2-sulfatase, alpha-N-acetyl glucosaminidase, and beta-glucuronidase are found. Conclusion: this biochemical method could potentially improve early diagnosis. Proteomic data supporting these results reveal disrupted biochemical pathways, including the degradation of dermatan sulfate, heparan sulfate, and cellular cholesterol trafficking. Full article

Lysosomes are essential intracellular organelles involved in plentiful cellular processes such as cell signaling, metabolism, growth, apoptosis, autophagy, protein processing, and maintaining cellular homeostasis. Their dysfunction is linked to various diseases, including lysosomal storage disorders, inflammation, cancer, cardiovascular diseases, neurodegenerative conditions, and aging. This review focuses on current and emerging therapies for lysosomal diseases (LDs), including small medicines, enzyme replacement therapy (ERT), gene therapy, transplantation, and lysosomal drug targeting (LDT). This study was conducted through databases like PubMed, Google Scholar, Science Direct, and other research engines. To treat LDs, medicines target the lysosomal membrane, acidification processes, cathepsins, calcium signaling, mTOR, and autophagy. Moreover, small-molecule therapies using chaperones, macro-therapies like ERT, gene therapy, and gene editing technologies are used as therapy for LDs. Additionally, endosymbiotic therapy, artificial lysosomes, and lysosomal transplantation are promising options for LD management. LDT enhances the therapeutic outcomes in LDs. Extracellular vesicles and mannose-6-phosphate-tagged nanocarriers display promising approaches for improving LDT. This study concluded that lysosomes play a crucial role in the pathophysiology of numerous diseases. Thus, restoring lysosomal function is essential for treating a wide range of conditions. Despite endosymbiotic therapy, artificial lysosomes, lysosomal transplantation, and LDT offering significant potential for LD control, there are ample challenges regarding safety and ethical implications. Full article

Phosphoglucomutase (EC 5.4.2.2., PGM), a key enzyme of glycogenolysis and glycogenesis, catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate, whereas phosphomannomutase (EC 5.4.2.8., PMM) transfers the phosphate group from the 1′ to the 6′, or from the 6′ to the 1′ position in mannose phosphate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, a single gene, Tk1108, encodes a protein with both PGM and PMM activities. Here, we report biophysical analysis and the 2.45 Å resolution cryo-EM structure of this novel enzyme. Our results demonstrate a specific arrangement of the four subunits in the quaternary structure, displaying a distinct catalytic cleft required for the bifunctional activity at extremely high temperatures. To the best of our knowledge, this is the first biophysical characterization and cryo-EM structure elucidation of a thermostable, bifunctional PGM/PMM. Full article

Background: Saline–alkali stress is a major factor limiting the growth of oats. Sugar is the primary carbon and energy source in plants which regulates plant development and growth by regulating enzyme activity and gene expression. Sucrose, glucose, and fructose are ubiquitous plant-soluble sugars that act as signalling molecules in the transcriptional regulation of various metabolic and defence-related genes. Methods: In this study, soluble sugars, fructose, sucrose, and starch contents were measured, and transcriptomics was used to determine the differentially expressed genes (DEGs) in saline-sensitive and saline-tolerant oats after 6, 12, 24, and 48 h. DEGs annotated to carbohydrates were selected using the Kyoto Encyclopedia of Genes and Genomes. Results: DEGs involved in carbohydrate metabolism were mainly enriched in the glycolysis/gluconeogenesis and pentose phosphate pathways, fructose and mannose metabolism, and starch and sucrose metabolism. GAPDH, SUPI, SUS2, ATP-PEK, HXK6, FBA4, TBA4, TKT, ISA3, PPDK1, and BAM2 were significantly expressed, and a quantitative reverse transcription polymerase chain reaction verified the transcriptome sequencing results. Conclusions: In this study, oats with different salinity tolerances were used to determine sugar contents under four salinity stress durations, and transcriptome sequencing was used to explore the regulatory mechanism of sugars and provide a reference for elucidating the sugar signalling regulatory mechanism under abiotic stress. Full article

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in hundreds of millions of infections and millions of deaths globally. Although vaccination campaigns are mitigating the pandemic, emerging viral variants continue to pose challenges. The spike (S) protein of SARS-CoV-2 plays a critical role in viral entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, making both proteins essential targets for therapeutic and vaccine development. The glycosylation of these proteins influences their structure and function. This underscores the need for detailed site-specific glycoproteomic analysis. In this study, we characterized the N- or O-glycosylation profiles of the recombinant receptor-binding domain (RBD) of spike protein and ACE2 proteins expressed from Expi293F cells, as well as the S2 subunit of spike protein expressed in plant (N. benthamiana) cells. Using a high-resolution Orbitrap Eclipse Tribrid mass spectrometer equipped with the Ultimate 3000 RSLCnano and I-GPA (Integrated GlycoProteome Analyzer) developed in a previous study, 148 N- and 28 O-glycopeptides from RBD, 71 N-glycopeptides from the S2 subunit, and 139 N-glycopeptides from ACE2 were characterized. In addition, we report post-translational modifications (PTMs) of glycan, including mannose-6-phosphate (M6P) and GlcNAc-1-phosphate-6-O-mannose in N-glycan of RBD and ACE2, and O-acetylation in O-glycan of RBD, identified for the first time in these recombinant proteins. The relative abundance distribution according to glycosites and glycan types were analyzed by quantified site-specific N- and O (only from RBD)-glycopeptides from RBD, S2, and ACE2 using I-GPA. Asn331 for RBD, Asn1098 for S2, and Asn103 for ACE2 were majorly N-glycosylated, and dominant glycan-type was complex from RBD and ACE2 and high-mannose from S2. These findings will provide valuable insights into the glycosylation patterns that influence protein function and immunogenicity and offer new perspectives for the development of vaccines and antibody-based therapies against COVID-19. Full article

Seed shattering (SS) functions are a survival mechanism in plants, enabling them to withstand adverse environmental conditions and ensure reproduction. However, this trait limits seed yield. Psathyrostachys juncea, a perennial forage grass with many favorable traits, is constrained by SS, limiting its broader application. To investigate the mechanisms underlying SS, second-generation Illumina sequencing and third-generation PacBio sequencing were conducted on abscission zone tissues of P. juncea at 7, 14, 21, and 28 days after heading. GO enrichment analysis identified several significant biological processes, including the “cell wall macromolecule catabolic process”, “cell wall polysaccharide catabolic process”, “hemicellulose catabolic process”, and “xylan catabolic process”, all involved in cell wall degradation. KEGG enrichment analysis showed that differentially expressed genes were predominantly enriched in pathways related to “starch and sucrose metabolism”, “fructose and mannose metabolism”, “phenylpropanoid biosynthesis”, “pentose and glucuronate interconversions”, and “galactose metabolism”, each linked to both the synthesis and degradation of the cell wall. Further analysis of the “starch and sucrose metabolism” pathway revealed genes encoding fructokinase, hexokinase, β-glucosidase, sucrose phosphate synthase, sucrose synthase, and endoglucanase, all of which affected cellulose content. Reduced cellulose content can alter cell wall structure, leading to SS. These findings provide new insights into the regulation of SS in P. juncea and offer valuable references for other species within the Poaceae family. Full article

Meat quality is a complex trait that exhibits significant variation across pig breeds, and the regulatory mechanisms governing pork meat quality are not fully elucidated. We compared the transcriptomics and metabolomics of the longissimus dorsi (LD) muscle between the Songliao Black Pig (SBP) and Large White × Landrace Pig (LWLDP) to investigate breed-specific differences in meat quality and underlying regulatory pathways. The results showed that SBP meat had a higher marbling score and backfat thickness, a richer color, a lower shear force, and reduced drip loss. Fatty acid (FA) analysis identified 15 significant FAs in the LWLDP, with docosahexaenoic acid (DHA) in the SBP, while amino acid (AA) analysis revealed no breed-based differences. Transcriptome analysis identified 134 upregulated and 362 downregulated genes in the SBP. Protein–protein interaction (PPI) network analysis found 25 key genes, which are associated with muscle development, fat deposition, and overall meat quality, while genes in the insulin signaling pathway, such as PPP1R3B, PPARGC1A, SOCS1, EIF4E, PRKAR2A, PRKAG2, and FASN, play a crucial role in balancing fat metabolism and catabolism. Metabolomic analysis identified 89 upregulated and 10 downregulated metabolites in the SBP, primarily involved in fructose and mannose metabolism, amino acid biosynthesis, nucleotide sugar metabolism, and glucagon signaling pathways. Gene–metabolite association analysis found that the PPP1R3B gene had a strong association with Thr-Leu, Maltol, D-myo-Inositol-4-phosphate, and Fructose-6-phosphate, while MYOG correlated with Mannose-6-phosphate, Fructose-1-phosphate, Mannose-1-phosphate, and Glucose-6-phosphate. In contrast, NR4A3 and PPARGC1A showed a strong negative correlation with most upregulated metabolites. In conclusion, this study identified functional genes, elucidated the mechanisms associated with meat quality traits, and identified gene–metabolite associations involved in energy metabolism, muscle development, and fat deposition, providing valuable insights into the molecular mechanisms that regulate meat quality between pig breeds. Full article

Mitochondrial DNA (mtDNA) supplementation can rescue poor oocyte quality and overcome embryonic arrest. Here, we investigated a series of sexually mature pigs generated through autologous and heterologous mtDNA supplementation. Brain, liver and heart tissues underwent metabolite profiling using gas chromatography–mass spectrometry and gene expression analysis through RNA-seq. They were then assessed for mRNA–metabolite interactions. The comparison between overall mtDNA supplemented and control pigs revealed that mtDNA supplementation reduced the lipids stearic acid and elaidic acid in heart tissue. However, heterologous mtDNA supplemented-derived pigs exhibited lower levels of abundance of metabolites when compared with autologous-derived pigs. In the brain, these included mannose, mannose 6-phosphate and fructose 6-phosphate. In the liver, maltose and cellobiose, and in the heart, glycine and glutamate were affected. mRNA–metabolite pathway analysis revealed a correlation between malate and CS, ACLY, IDH2 and PKLR in the liver and glutamate and PSAT1, PHGDH, CDO1 and ANPEP in the heart. Our outcomes demonstrate that mtDNA supplementation, especially heterologous supplementation, alters the metabolite and transcriptome profiles of brain, liver, and heart tissues. This is likely due to the extensive resetting of the balance between the nuclear and mitochondrial genomes in the preimplantation embryo, which induces a series of downstream effects. Full article