Protein Synthesis In Cells Research Articles

Pathogenic Mechanisms of DDX41 Mutations in the Development of Myeloid Malignancies

DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele (most commonly p.A500fs or p.D140fs, depending on the ethnicity) is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms have not fully been elucidated. To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles. Vav1-Cre mediated homozygous deletion of Ddx41 resulted in embryonic lethality, suggesting that Ddx41 is indispensable for normal hematopoiesis. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 ( Ddx41 +/+), heterozygous Ddx41 KO( Ddx41 +/-), homozygous Ddx41 KO( Ddx41 -/-), heterozygous for the Ddx41 R525H mutation ( Ddx41 R525H/+), or hemizygous for the Ddx41 R525H mutation( Ddx41 R525H/-), in which expression of the mutant allele was induced by tamoxifen administration. First, we assessed cell intrinsic effects of these Ddx41 alleles, using noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were transplanted with BM from Ddx41 -/- or Ddx41 R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF), which was not observed in mice transplanted with BM from Ddx41 +/+, Ddx41 +/- or Ddx41 R525H/+ mice. By contrast, the mice transplanted with Ddx41 +/- or Ddx41 R525H/+ BM showed significantly reduced WBC counts and anemia in long-term observation in both primary and serial transplantations. Some of the Ddx41 +/- or Ddx41 R525H/+ BM-transplanted mice exhibited MDS-like phenotypes, showing ineffective hematopoiesis with evidence of erythroid dysplasia. Transcriptome analysis revealed that stem cells (Kit +Sca-1 -Lin low cells) derived from Ddx41 R525H/- BM-transplanted mice exhibited a significant upregulation of genes involved in innate immunity, including interferon stimulated genes, compared with stem cells derived from Ddx41 +/+ BM-transplanted mice. In addition, snoRNA and ribosomal genes were significantly deregulated in stem cells from Ddx41 -/- and Ddx41 R525H/- BM-transplanted mice, which could result in abnormal ribosome biogenesis and protein synthesis in Ddx41 mutant cells. We also assessed the reconstitution capacity of whole BM cells from different Ddx41 mutant mice in competitive transplantation experiments. The donor chimerism of Ddx41 -/- and Ddx41 R525H/- BM-transplanted mice in PB was markedly reduced compared to Ddx41 +/+ BM-transplanted mice. In contrast, Ddx41 +/- and Ddx41 R525H/+ BM-transplanted micedid not show significant changes in competitive bone marrow reconstitution compared to Ddx41 +/+ BM-transplanted mice. Given that the MDS clones bearing DDX41 R525H somatic alleles are typically observed as a small subclone in patients, we next co-transplanted Ddx41 +/+- and Ddx41 525H/--derived BM cells with Ddx41 +/+- or Ddx41 +/--derived BM cells at the ratio of 1:9. Recipient mice showed significantly reduced WBC counts when Ddx41 +/+- or Ddx41 +/- were co-transplanted with Ddx41 525H/- -derived BM, suggesting that Ddx41 525H/--derived hematopoietic cells have negative effect on normal hematopoiesis. In summary, compound biallelic loss-of function and R525 alleles led to severe BM failure. Monoallelic Ddx41 loss-of function and R525H knock-in alleles, by contrast, are compatible with hematopoiesis but associated with an impairment of hematopoiesis and the development of MDS with ageing, where activated innate immunity, impaired RNA metabolism and ribosome functions may play important roles.

A review of photobiomodulation on PI3K/AKT/mTOR in wound healing

Wound healing involves a series of cellular and molecular processes to heal injured tissue. Growth factors such as vascular endothelial growth factor (VEGF), and signalling pathways such as phosphatidylinositol 3-kinase, protein kinase B, and mammalian target of rapamycin (PI3K/AKT/mTOR) are essential in wound healing. VEGF is linked to intracellular signalling pathways including PI3K/AKT/mTOR, which controls cell growth, metabolism, proliferation, apoptosis, and protein synthesis. During photobiomodulation (PBM), low-level light in the visible red and near-infrared (NIR) spectrum is employed to promote healing, and reduce pain, inflammation, and oedema. Several studies demonstrate that PBM enhances cellular survival, proliferation, migration, and viability in vitro, however, the exact cellular and molecular mechanisms responsible for these benefits have not yet been identified. The aim of this review is to explore the effects of PBM on the PI3K/AKT/mTOR signalling pathway in wound healing.

Serine synthesis pathway enzyme PHGDH is critical for muscle cell biomass, anabolic metabolism, and mTORC1 signaling.

Cells use glycolytic intermediates for anabolism, e.g., via the serine synthesis and pentose phosphate pathways. However, we still understand poorly how these metabolic pathways contribute to skeletal muscle cell biomass generation. The first aim of this study was therefore to identify enzymes that limit protein synthesis, myotube size, and proliferation in skeletal muscle cells. We inhibited key enzymes of glycolysis, the pentose phosphate pathway, and the serine synthesis pathway to evaluate their importance in C2C12 myotube protein synthesis. Based on the results of this first screen, we then focused on the serine synthesis pathway enzyme phosphoglycerate dehydrogenase (PHGDH). We used two different PHGDH inhibitors and mouse C2C12 and human primary muscle cells to study the importance and function of PHGDH. Both myoblasts and myotubes incorporated glucose-derived carbon into proteins, RNA, and lipids, and we showed that PHGDH is essential in these processes. PHGDH inhibition decreased protein synthesis, myotube size, and myoblast proliferation without cytotoxic effects. The decreased protein synthesis in response to PHGDH inhibition appears to occur mainly mechanistic target of rapamycin complex 1 (mTORC1)-dependently, as was evident from experiments with insulin-like growth factor 1 and rapamycin. Further metabolomics analyses revealed that PHGDH inhibition accelerated glycolysis and altered amino acid, nucleotide, and lipid metabolism. Finally, we found that supplementing an antioxidant and redox modulator, N-acetylcysteine, partially rescued the decreased protein synthesis and mTORC1 signaling during PHGDH inhibition. The data suggest that PHGDH activity is critical for skeletal muscle cell biomass generation from glucose and that it regulates protein synthesis and mTORC1 signaling.NEW & NOTEWORTHY The use of glycolytic intermediates for anabolism was demonstrated in both myoblasts and myotubes, which incorporate glucose-derived carbon into proteins, RNA, and lipids. We identify phosphoglycerate dehydrogenase (PHGDH) as a critical enzyme in those processes and also for muscle cell hypertrophy, proliferation, protein synthesis, and mTORC1 signaling. Our results thus suggest that PHGDH in skeletal muscle is more than just a serine-synthesizing enzyme.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature.Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry.Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, “pB513-b CMV-P/4shRNAs/GFP” plasmid, “pB513-bHSP90-P/4sh-RNAs/GFP” and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days.Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

Methionine Promotes Milk Protein Synthesis via the PI3K-mTOR Signaling Pathway in Human Mammary Epithelial Cells.

Breast milk is widely considered to be the most natural, safe, and complete food for infants. However, current breastfeeding rates fall short of the recommendations established by the World Health Organization. Despite this, there are few studies that have focused on the promotion of human lactation through nutrient supplementation. Therefore, the aim of this study was to investigate the effect of methionine on milk synthesis in human mammary epithelial cells (MCF-10A cells) and to explore the underlying mechanisms. To achieve this, MCF-10A cells were cultured with varying concentrations of methionine, ranging from 0 to 1.2 mM. Our results indicated that 0.6 mM of methionine significantly promoted the synthesis of milk protein. An RNA-seq analysis revealed that methionine acted through the PI3K pathway. This finding was validated through real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. In addition, PI3K inhibition assays confirmed that methionine upregulated the expression of both mTOR and p-mTOR through activation of PI3K. Taken together, these findings suggest that methionine positively regulates milk protein synthesis in MCF-10A cells through the PI3K-mTOR signaling pathway.

Association Between Patient Characteristics and Histopathological Features of Lung Cancer At RSUP Dr. Mohammad Hoesin Palembang

Introduction. Lung cancer is the uncontrolled growth of malignant cells by the body in lung tissue and is caused by genetic mutations and changes in protein synthesis in normal cells. Histopathological features of lung cancer include squamous cell carcinoma, adenocarcinoma, small cell carcinoma, and other types. Early detection, as well as prevention, needs to be done in high-risk communities with the aim of reducing the number of events. This study aims to determine the relationship between sociodemographic factors and histopathological features of lung cancer. Methods. This study is an analytic observational study with a cross-sectional design. The research sample is in the form of secondary data from the Department of Anatomical Pathology and Medical Record Installation for 2019-2021. The research was conducted at Dr. Mohammad Hoesin Palembang. The sampling technique was carried out by total sampling. Results. Most of the respondents were in the age group >45 years (84.1%), male (78.3%), had no history of smoking (60.9%), the predominant main complaint was shortness of breath (63, 8%), the most common histopathological picture is adenocarcinoma. There was no significant relationship between age (p=0.998), gender (p=0.736), smoking history (p=0.572) and histopathological features of lung cancer patients. Conclusion. There was no significant relationship between age, sex and smoking status on the histopathological features of lung cancer.

Genetic Determinants of Dependency on Regulated Protein Synthesis in Leukemia Stem Cells

Acute myeloid leukemia (AML) is initiated and sustained by rare self-renewing leukemia stem cells (LSCs). LSCs do not resemble HSCs, but rather more mature hematopoietic progenitor cells that usually have no self-renewal potential. However, during leukemogenesis, LSCs become aberrantly self-renewing, but the mechanisms that allow for this are poorly understood. We recently used models of AML driven by AML-ETO9a and MLL-AF9 to show that LSCs may co-opt from HSCs a dependence on regulation of protein synthesis mediated by CD99 (ASH 2021, #2222), which we identified as a therapeutic target highly expressed in LSCs (PMID: 28123069). However, it remains unclear if LSCs from specific subtypes of AML are particularly dependent on regulated protein synthesis. In an analysis of transcriptome data from the ECOG 1900 cohort, we found that certain mutations ( KIT, PTEN, AML-ETO, IDH1/2 and FLT3-ITD) were associated with the highest levels of CD99, raising the possibility that such AML subtypes are more dependent on CD99. In the TCGA AML cohort, CD99 expression was significantly higher in AML patients with FLT3-ITD mutations but not NRAS mutations ( Fig. 1A). To test if LSCs from AMLs driven by mutations in FLT3 or NRAS differ in their dependence on regulation of protein synthesis by CD99, we crossed Cd99 -/-(CD99 KO) mice with two mouse models of AML driven by these alleles to generate Cd99 -/-; Mx1-Cre; Tet2 fl/fl; Flt3 ITD/+and Cd99 -/-; Mx1-Cre; Tet2 fl/fl; Nras LSL-G12D/+ mice (CD99 KO TET2/FLT3 and CD99 KO TET2/NRAS). TET2 was chosen as a cooperating allele given its neutral association with CD99 in the above human datasets, and it additionally co-occurs frequently with mutations in FLT3 or NRAS. To generate AML in both models, Tet2 was deleted with polyinosine-polycytidylic acid injections, followed by transplant of 4 million bone marrow (BM) cells into irradiated wild-type (WT) mice. At 16 weeks, recipients of CD99 WT TET2/FLT3 BM developed an expansion of aberrant donor-derived Mac-1 + c-kit + cells in the peripheral blood (PB), and BM aspirates revealed an accumulation of LSK CD48 +CD150 - cells that harbor LSC activity in this model ( Fig. 1B). In contrast, recipients of CD99 KO TET2/FLT3 BM exhibited significant depletion of these aberrant populations (P=0.0055 and P=0.019 respectively, n=10 for each group). Recipients of CD99 WT TET2/FLT3 BM also exhibited increased leukocytosis and thrombocytopenia, which were attenuated in recipients of CD99 KO TET2/FLT3 BM (P=0.046 and P=0.042, respectively). In contrast, there was no difference between recipients of CD99 WT TET2/NRAS and CD99 KO TET2/NRAS BM in emergence of the aberrant Mac-1 + c-kit + cells and thrombocytopenia (P=0.3237 and P=0.9752, respectively, n=10 for each group). These data suggest that loss of CD99 may impair leukemogenesis in TET2/FLT3 but not TET2/NRAS AMLs. To determine if CD99 is required for LSC function, we performed secondary transplants of one million BM cells from primary recipients into lethally irradiated WT recipients. Mice transplanted with CD99 KO TET2/FLT3 BM, compared with those transplanted with CD99 WT TET2/FLT3 BM, demonstrated fewer donor-derived cells in the PB. While CD99 WT TET2/FLT3-transplanted mice succumbed to AML at a median of 11 weeks, all CD99 KO TET2/FLT3-transplanted mice survived beyond 16 weeks. PB smears from CD99 KO TET2/FLT3 mice demonstrated leukocytes with condensed chromatin and lobulated nuclei, indicative of restored hematopoietic maturation. Finally, in vivo O-propargyl-puromycin assays showed that loss of CD99 resulted in increased protein synthesis in LSCs (LSK CD48 +CD150 -) (fold-change=1.69±0.18, n=3 for each group). In contrast, mice secondarily transplanted with CD99 KO TET2/NRAS BM exhibited similar engraftment, survival, and morphologic evidence of AML as those transplanted with CD99 WT TET2/NRAS BM. In conclusion, our data demonstrate that LSC function in AML driven by mutated FLT3, but not mutated NRAS, is dependent on maintenance of regulated protein synthesis by CD99. We speculate that this differential dependency may reflect a different cell-of-origin, as recent work has shown that AMLs associated with FLT3-ITD exhibit particularly primitive cellular hierarchies (PMID: 35618837). Finally, our results suggest that certain high-risk AML sub-types associated with elevated CD99 expression may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs.

Recent Advances in mRNA Vaccine Development

Messenger RNA vaccines are vaccine that utilizes a small segment of genetic material, messenger RNA (mRNA), to provide instructions to cells to produce a specific protein. This mRNA is synthesized in a laboratory and packaged into lipid nanoparticles, which protect and facilitate its entry into cells for protein synthesis. Upon injection into the muscle of the recipient, the mRNA instructs cells to produce a protein that is displayed on the surface of the cell, triggering an immune response. The immune system then produces antibodies and activates immune cells to target and eliminate the protein, while also generating memory cells to respond quickly in the event of future pathogen encounters. Available mRNA vaccines, such as Pfizer-BioNTech and Moderna, were developed and authorized for emergency use within a year. These vaccines require extensive cold chain storage, antigen delivery, potential immune response variability optimization, and sophisticated manufacturing process. To improve their effectiveness, stability, and delivery, efforts are underway to explore next-generation mRNA vaccines. Research is focused on enhancing the stability of mRNA vaccines, particularly their temperature sensitivity, to facilitate easier storage and distribution. Self-amplifying mRNA vaccines are also being developed to generate multiple copies of mRNA within cells, potentially leading to a higher production of protein and a stronger immune response. Studies are also exploring new delivery systems using specialized nanoparticles and liposomes to specifically target certain immune cells. Additionally, the development of combination vaccines, including multiple mRNA sequences in single vaccine, is being investigated to protect against multiple strains or variants of particular pathogen simultaneously. Direct delivery of mRNA vaccines into the skin is being explored as a means of enhancing immune response and reducing the required vaccine dose. In summary, messenger RNA vaccines represent a promising new approach to vaccination, with ongoing research aimed at improving their effectiveness, stability, and delivery

193 Creatine Monohydrate on In Vitro Cultivated Bovine Cells Leading to Guanidinoacetic Acid Supplementation In Vivo

Abstract Guanidinoacetic acid (GAA) is an amino acid-like compound that acts as a precursor to creatine (CRE) when either made in the body or digested. Creatine is directly used by skeletal muscle tissue as an energy source with the potential to act as a growth-promoting agent. The study objectives were to evaluate the effects of various CRE concentrations on bovine satellite cells for the rate of proliferation and myogenic differentiation. Satellite cells (SC) were isolated from the Longissimus muscle of 3-month-old Holstein bulls (n = 5). Cells were seeded at a density of 1 × 104 cells/mL on 96-well plates and incubated with 0 (CON), 2.5, 5, 7.5, and 10 mM of CRE. The absorbance rate was measured at 0, 24, 48, 72, and 96 h of incubation. Cells for gene expression were plated on 6-well plates at a density of 1 × 105 in 10% FBS/DMEM. Following 24 h of adhesion, SC were incubated with 5 and 7.5mM CRE for 48 h. After which, cells were harvested after 48 h for Pax3, Pax7, and Myf5 mRNA gene expression analysis. To identify the effects of CRE during myogenic differentiation, SCs were induced myogenic differentiation. Upon reaching approximately 90% confluency, the growth medium was replaced by differentiation media (2% horse serum/ DMEM) containing 0 (CON), 5, and 7.5 mM CRE. A two-way ANOVA and Tukey's HSD test were conducted regarding cell proliferation. A one-way ANOVA and Tukey's HSD test were performed for gene expression. Interaction of treatment against time was noted (P < 0.01) in Con, 2.5, 5, 7.5, and 10 mM CRE. Cell proliferation rate was influenced by CRE treatment (P < 0.01). At 72 h of incubation, all CRE treatments increased cell proliferation rate compared with Con (P < 0.05). At 96 h of incubation, cells treated with 2.5, 5, and 7.5 mM of CRE increased proliferation compared with Con (P < 0.05). At 96 h, CRE treatment 2.5, 5, and 7.5 mM increased proliferation rate compared with 10mM CRE treated cells (P < 0.05). No treatment effect was found in the mRNA gene expression of Pax7, Pax3, and Myf5 (P > 0.05). Cells incubated with CRE showed no change in mRNA gene expression of IGF-1 and Myogenin (P > 0.05), and a tendency was noted for S6KB1 increase expression under treatment (P = 0.0643). S6KB1 is a protein kinase that, through mTOR pathway expression, leads to an increase in cell growth, protein synthesis, and cell proliferation. Results suggest that the treatment of BSCs with CRE impacted proliferation, CRE at 5mM vs. 7.5 mM concentrations treatment of differentiated muscle cells led to a trend in altering S6KB1 gene expression.

Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells’ molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as “adaptive” (ADA) or “non-adaptive” (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor’s ability to survive. Depending on the tumor’s adaptability potential, subpopulations with acquired resistance mechanisms may arise.

Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

Mechanism and evolutionary analysis of Yarrowia lipolytica CA20 capable of producing erythritol with a high yield based on comparative genomics.

Combined mutagenesis is widely applied for the breeding of robust Yarrowia lipolytica used in the production of erythritol. However, the changes of genome after mutagenesis remains unclear. This study aimed to unravel the mechanism involved in the improved erythritol synthesis of CA20 and the evolutionary relationship between different Y. lipolytica by comparative genomics analysis. The results showed that the genome size of Y. lipolytica CA20 was 20,420,510 bp, with a GC content of 48.97%. There were 6330 CDS and 649 ncRNA (non-coding RNA) in CA20 genome. Average nucleotide identity (ANI) analysis showed that CA20 genome possessed high similarity (ANI > 99.50%) with other Y. lipolytica strains, while phylogenetic analysis displayed that CA20 was classified together with Y. lipolytica IBT 446 and Y. lipolytica H222. CA20 shared 5342 core orthologous genes with the 8 strains while harbored 65 specific genes that mainly participated in the substrate and protein transport processes. CA20 contained 166 genes coding for carbohydrate-active enzymes (CAZymes), which was more than that found in other strains (108－137). Notably, 4, 2, and 13 different enzymes belonging to glycoside hydrolases (GHs), glycosyltransferases (GTs), and carbohydrate esterases (CEs), respectively, were only found in CA20. The enzymes involved in the metabolic pathway of erythritol were highly conserved in Y. lipolytica, except for transaldolase (TAL1). In addition, the titer and productivity of erythritol by CA20 were 190.97 g/L and 1.33 g/L/h, respectively, which were significantly higher than that of WT5 wherein 128.61 g/L and 0.92 g/L/h were obtained (P< 0.001). Five frameshift mutation genes and 15 genes harboring nonsynonymous mutation were found in CA20 compared with that of WT5. Most of these genes were involved in the cell division, cell wall synthesis, protein synthesis, and protein homeostasis maintenance. These findings suggested that the genome of Y. lipolytica is conserved during evolution, and the variance of living environment is one important factor leading to genome divergence. The varied number of CAZymes existed in Y. lipolytica is one factor that contributes to the performance difference. The increased synthesis of erythritol by Y. lipolytica CA20 is correlated with the improvement of the stability of cell structure and internal environment. The results of this study provide a basis for the directional breeding of robust strains used in erythritol production.

Enhancement of PPARα-Inhibited Leucine Metabolism-Stimulated β-Casein Synthesis and Fatty Acid Synthesis in Primary Bovine Mammary Epithelial Cells.

Leucine, a kind of branched-chain amino acid, plays a regulatory role in the milk production of mammalian mammary glands, but its regulatory functions and underlying molecular mechanisms remain unknown. This work showed that a leucine-enriched mixture (LEUem) supplementation increased the levels of milk protein and milk fat synthesis in primary bovine mammary epithelial cells (BMECs). RNA-seq of leucine-treated BMECs indicated alterations in lipid metabolism, translation, ribosomal structure and biogenesis, and inflammatory response signaling pathways. Meanwhile, the supplementation of leucine resulted in mTOR activation and increased the expression of BCKDHA, FASN, ACC, and SCD1. Interestingly, the expression of PPARα was independently correlated with the leucine-supplemented dose. PPARα activated by WY-14643 caused significant suppression of lipogenic genes expression. Furthermore, WY-14643 attenuated leucine-induced β-casein synthesis and enhanced the level of BCKDHA expression. Moreover, promoter analysis revealed a peroxisome-proliferator-response element (PPRE) site in the bovine BCKDHA promoter, and WY-14643 promoted the recruitment of PPARα onto the BCKDHA promoter. Together, the present data indicate that leucine promotes the synthesis of β-casein and fatty acid and that PPARα-involved leucine catabolism is the key target.

Obtaining New Candidate Peptides for Biological Anticancer Drugs from Enzymatic Hydrolysis of Human and Bovine Hemoglobin.

Enzymatic hydrolysis of bovine and human hemoglobin generates a diversity of bioactive peptides, mainly recognized for their antimicrobial properties. However, antimicrobial peptides stand out for their ability to specifically target cancer cells while preserving rapidly proliferating healthy cells. This study focuses on the production of bioactive peptides from hemoglobin and evaluates their anticancer potential using two distinct approaches. The first approach is based on the use of a rapid screening method aimed at blocking host cell protein synthesis to evaluate candidate anticancer peptides, using Lepidium sativum seed germination as an indicator. The results show that: (1) The degree of hydrolysis (DH) significantly influences the production of bioactive peptides. DH levels of 3 to 10% produce a considerably stronger inhibition of radicle growth than DH 0 (the native form of hemoglobin), with an intensity three to four times greater. (2) Certain peptide fractions of bovine hemoglobin have a higher activity than those of human hemoglobin. (3) The structural characteristics of peptides (random coil or alpha helix) play a crucial role in the biological effects observed. (4) The α137-141 peptide, the target of the study, was the most active of the fractions obtained from bovine hemoglobin (IC50 = 29 ± 1 µg/mL) and human hemoglobin (IC50 = 48 ± 2 µg/mL), proving to be 10 to 15 times more potent than the other hemoglobin fractions, attributed to its strong antimicrobial potential. The second approach to assessing anticancer activity is based on the preliminary in vitro analysis of hydrolysates and their peptide fractions, with a focus on the eL42 protein. This protein is of major interest due to its overexpression in all cancer cells, making it an attractive potential target for the development of anticancer molecules. With this in mind, astudy was undertaken using a method for labeling formylase (formyl-methionyl-tRNA transformylase (FMTS)) with oxidized tRNA. This approach was chosen because of the similarities in the interaction between formylase and the eL42 protein with oxidized tRNA. The results obtained not only confirmed the previous conclusions but also reinforced the hypothesis that the inhibition of protein synthesis plays a key role in the anticancer mechanism of these peptides. Indeed, the data suggest that samples containing α137-141 peptide (NKT) and total hydrolysates may have modulatory effects on the interaction between FMTS and oxidized tRNA. This observation highlights the possibility that the latter could influence molecular binding mechanisms, potentially resulting in a competitive situation where the ability of substrate tRNA to bind efficiently to ribosomal protein is compromised in their presence. Ultimately, these results suggest the feasibility of obtaining candidate peptides for biological anticancer drugs from both human and bovine hemoglobin sources. These scientific advances show new hope in the fight against cancer, which affects a large number of people around the world.

Increase CO2 recycling of Escherichia coli containing CBB genes by enhancing solubility of multiple expressed proteins from an operon through temperature reduction.

One of the current promising solutions to address problems of CO2 emissions is the development of biological carbon fixation strategies. This strategy leads to higher chemical biosynthetic productivity through improved biological CO2 fixation. In a previous study, we introduced the Calvin-Benson Bassham (CBB) genes of the photosynthetic bacterium Cereibacter sphaeroides into Escherichia coli to develop a strain capable of endogenous CO2 recycling by heterologous expression of the CBB genes. However, the heterologous expression of recombinant proteins in E. coli is often hampered by inclusion bodies (IB), which are protein aggregates. Various factors contribute to IB formation, including host cell metabolism, protein synthesis, transformation machinery, and target protein properties. In this study, we investigated the influence of environmental conditions, particularly culture temperature, on IB formation and CO2 recycling in the CBB strain. As a result, by reducing the culture temperature from 37°C to 30°C, a significant suppression of IB formation was achieved, resulting in a remarkable decrease in CO2 release by approximately 5.76 times. In addition, interestingly, an enhancement in the accumulation of pyruvate by approximately 2.3 times was observed at the same time. These results demonstrate the simultaneous improvements in CO2 recycling and the synthesis of organic acids achieved through temperature control. Based on the findings of this study, we believe that temperature control would be a promising approach to increasing chemical biosynthetic productivity as well as biological CO2 fixation activity in integrated autotrophic biorefinery strategies. IMPORTANCE In a previous study, we successfully engineered Escherichia coli capable of endogenous CO2 recycling through the heterologous expression of the Calvin-Benson Bassham genes. Establishing an efficient gene expression environment for recombinant strains is crucial, on par with the importance of metabolic engineering design. Therefore, the primary objective of this study was to further mitigate greenhouse gas emissions by investigating the effects of culture temperature on the formation of inclusion bodies (IB) and CO2 fixation activity in the engineered bacterial strain. The findings demonstrate that lowering the culture temperature effectively suppresses IB formation, enhances CO2 recycling, and concurrently increases the accumulation of organic acids. This temperature control approach, without adding or modifying compounds, is both convenient and efficient for enhancing CO2 recycling. As such, additional optimization of various environmental parameters holds promise for further enhancing the performance of recombinant strains efficiently.

Downregulation of stromal syntenin sustains AML development

The crosstalk between cancer and stromal cells plays a critical role in tumor progression. Syntenin is a small scaffold protein involved in the regulation of intercellular communication that is emerging as a target for cancer therapy. Here, we show that certain aggressive forms of acute myeloid leukemia (AML) reduce the expression of syntenin in bone marrow stromal cells (BMSC). Stromal syntenin deficiency, in turn, generates a pro‐tumoral microenvironment. From serial transplantations in mice and co‐culture experiments, we conclude that syntenin‐deficient BMSC stimulate AML aggressiveness by promoting AML cell survival and protein synthesis. This pro‐tumoral activity is supported by increased expression of endoglin, a classical marker of BMSC, which in trans stimulates AML translational activity. In short, our study reveals a vicious signaling loop potentially at the heart of AML–stroma crosstalk and unsuspected tumor‐suppressive effects of syntenin that need to be considered during systemic targeting of syntenin in cancer therapy.

Key transcriptional effectors of the pancreatic acinar phenotype and oncogenic transformation.

Proper maintenance of mature cellular phenotypes is essential for stable physiology, suppression of disease states, and resistance to oncogenic transformation. We describe the transcriptional regulatory roles of four key DNA-binding transcription factors (Ptf1a, Nr5a2, Foxa2 and Gata4) that sit at the top of a regulatory hierarchy controlling all aspects of a highly differentiated cell-type-the mature pancreatic acinar cell (PAC). Selective inactivation of Ptf1a, Nr5a2, Foxa2 and Gata4 individually in mouse adult PACs rapidly altered the transcriptome and differentiation status of PACs. The changes most emphatically included transcription of the genes for the secretory digestive enzymes (which conscript more than 90% of acinar cell protein synthesis), a potent anabolic metabolism that provides the energy and materials for protein synthesis, suppressed and properly balanced cellular replication, and susceptibility to transformation by oncogenic KrasG12D. The simultaneous inactivation of Foxa2 and Gata4 caused a greater-than-additive disruption of gene expression and uncovered their collaboration to maintain Ptf1a expression and control PAC replication. A measure of PAC dedifferentiation ranked the effects of the conditional knockouts as Foxa2+Gata4 > Ptf1a > Nr5a2 > Foxa2 > Gata4. Whereas the loss of Ptf1a or Nr5a2 greatly accelerated Kras-mediated transformation of mature acinar cells in vivo, the absence of Foxa2, Gata4, or Foxa2+Gata4 together blocked transformation completely, despite extensive dedifferentiation. A lack of correlation between PAC dedifferentiation and sensitivity to oncogenic KrasG12D negates the simple proposition that the level of differentiation determines acinar cell resistance to transformation.

Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins.

Human cytomegalovirus (HCMV) requires inactivation of AKT to efficiently replicate, yet how AKT is shut off during HCMV infection has remained unclear. We show that UL38, an HCMV protein that activates mTORC1, is necessary and sufficient to destabilize insulin receptor substrate 1 (IRS1), a model insulin receptor substrate (IRS) protein. Degradation of IRS proteins in settings of excessive mTORC1 activity is an important mechanism for insulin resistance. When IRS proteins are destabilized, PI3K cannot be recruited to growth factor receptor complexes, and hence, AKT membrane recruitment, a rate limiting step in its activation, fails to occur. Despite its penchant for remodeling host cell signaling pathways, our results reveal that HCMV relies upon a cell-intrinsic negative regulatory feedback loop to inactivate AKT. Given that pharmacological inhibition of PI3K/AKT potently induces HCMV reactivation from latency, our findings also imply that the expression of UL38 activity must be tightly regulated within latently infected cells to avoid spontaneous reactivation.

Gly-Leu instead of Gly promoted the proliferation and protein synthesis of chicken intestinal epithelial cells

Gly-Leu instead of Gly promoted the proliferation and protein synthesis of chicken intestinal epithelial cells

Cordycepin, a bioactive compound from Cordyceps spp., moderates Alzheimer’s disease-associated pathology via anti-oxidative stress and autophagy activation

Alzheimer’s causes cognitive dysfunction. This study investigated the neuro-promoting effects of cordycepin on amyloid-beta precursor protein (APP) synthesis in human neuroblastoma SH-SY5Y cells. Cordycepin was found to boost SH-SY5Y cell proliferation and decreased AD pathology. APP, PS1, and PS2 were downregulated whereas ADAM10 and SIRT1 were upregulated by cordycepin. Cordycepin also reduced APP secretion in a dose-dependent manner. Cordycepin alleviated oxidative stress by the upregulation of GPX and SOD, as well as autophagy genes (LC3, ATG5, and ATG12). Cordycepin activity was also found to be SIRT1-dependent. Therefore, cordycepin may relieve the neuronal degeneration caused by APP overproduction, and oxidative stress.