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Bioengineering (Basel, Switzerland) Mar 2024Due to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as...
Due to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high-seeding-density (HSD) strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells, focused on identifying significant metabolites and their interactions. We observed high glycolytic fluxes, the depletion of asparagine, and a shift from lactate production to consumption. Using a metabolic network and flux analysis, we compared the standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched with amino acids. We reconstructed a metabolic network and kinetic models based on the observations and explored the effects of different feeding strategies on CHO cell metabolism. Our findings revealed that the addition of a bolus medium (BM) containing asparagine improved final titers. However, increasing the asparagine concentration in the feed further prevented the lactate shift, indicating a need to find a balance between increased asparagine to counteract limitations and lower asparagine to preserve the shift in lactate metabolism.
PubMed: 38671753
DOI: 10.3390/bioengineering11040331 -
Metabolites Apr 2024Sugarcane ( spp. hybrids) and its processed products have supported local industries such as those in the Nansei Islands, Japan. To improve the sugarcane quality and...
Sugarcane ( spp. hybrids) and its processed products have supported local industries such as those in the Nansei Islands, Japan. To improve the sugarcane quality and productivity, breeders select better clones by evaluating agronomic characteristics, such as commercially recoverable sugar and cane yield. However, other constituents in sugarcane remain largely unutilized in sugarcane breeding programs. This study aims to establish a data-driven approach to analyze agronomic characteristics from breeding programs. This approach also determines a correlation between agronomic characteristics and free amino acid composition to make breeding programs more efficient. Sugarcane was sampled in clones in the later stage of breeding selection and cultivars from experimental fields on Tanegashima Island. Principal component analysis and hierarchical cluster analysis using agronomic characteristics revealed the diversity and variability of each sample, and the data-driven approach classified cultivars and clones into three groups based on yield type. A comparison of free amino acid constituents between these groups revealed significant differences in amino acids such as asparagine and glutamine. This approach dealing with a large volume of data on agronomic characteristics will be useful for assessing the characteristics of potential clones under selection and accelerating breeding programs.
PubMed: 38668371
DOI: 10.3390/metabo14040243 -
Journal of Animal Science Jan 2024Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes endocrine disruption and porcine reproductive dysfunction. Heat stress (HS) occurs when exogenous and...
Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes endocrine disruption and porcine reproductive dysfunction. Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation. Independently, HS and ZEN both compromise swine reproduction; thus, the hypothesis investigated was two-pronged: that ZEN exposure would alter the ovarian proteome and that these effects would differ in thermal neutral (TN) and HS pigs. Pre-pubertal gilts (n = 38) were fed ad libitum and assigned to either (TN: 21.0 ± 0.1 °C) or HS (12 h cyclic temperatures of 35.0 ± 0.2 °C and 32.2 ± 0.1 °C). Within the TN group, a subset of pigs were pair-fed (PF) to the amount of feed that the HS gilts consumed to eliminate the confounding effects of dissimilar nutrient intake. All gilts orally received a vehicle control (CT) or ZEN (40 μg/kg/BW) resulting in six treatment groups: thermoneutral (TN) vehicle control (TC; n = 6); TN ZEN (TZ; n = 6); PF vehicle control (PC; n = 6); PF ZEN (PZ; n = 6); HS vehicle control (HC; n = 7); or HS ZEN (HZ; n = 7) for 7 d. When compared to the TC pigs, TZ pigs had 45 increased and 39 decreased proteins (P ≤ 0.05). In the HZ pigs, 47 proteins were increased and 61 were decreased (P ≤ 0.05). Exposure to ZEN during TN conditions altered sec61 translocon complex (40%), rough endoplasmic reticulum membrane (8.2%), and proteasome complex (5.4%), asparagine metabolic process (0.60%), aspartate family amino acid metabolic process (0.14%), and cellular amide metabolic process (0.02%) pathways. During HS, ZEN affected cellular pathways associated with proteasome core complex alpha subunit complex (0.23%), fibrillar collagen trimer (0.14%), proteasome complex (0.05%), and spliceosomal complex (0.03%). Thus, these data identify ovarian pathways altered by ZEN exposure and suggest that the molecular targets of ZEN differ in TN and HS pigs.
Topics: Animals; Zearalenone; Female; Ovary; Proteome; Swine; Hot Temperature; Heat-Shock Response; Estrogens, Non-Steroidal
PubMed: 38666409
DOI: 10.1093/jas/skae115 -
IScience May 2024Mounting evidence supports the role of neuroinflammation in radiation-induced brain injury (RIBI), a chronic disease characterized by delayed and progressive...
Mounting evidence supports the role of neuroinflammation in radiation-induced brain injury (RIBI), a chronic disease characterized by delayed and progressive neurological impairment. Asparagine endopeptidase (AEP), also known as legumain (LGMN), participates in multiple malignancies and neurodegenerative diseases and may potentially be involved in RIBI. Here, we found AEP expression was substantially elevated in the cortex and hippocampus of wild-type () mice following whole-brain irradiation. knockout () alleviated neurological impairment caused by whole-brain irradiation by suppressing neuronal senescence. Bulk RNA and metabolomic sequencing revealed AEP's involvement in the antigen processing and presentation pathway and neuroinflammation. This was further confirmed by co-culturing primary neurons with the conditioned media derived from irradiated or primary microglia. Furthermore, esomeprazole inhibited the enzymatic activity of AEP and RIBI. These findings identified AEP as a critical factor of neuroinflammation in RIBI, highlighting the prospect of targeting AEP as a therapeutic approach.
PubMed: 38655198
DOI: 10.1016/j.isci.2024.109698 -
Communications Biology Apr 2024NGLY1 deficiency is a genetic disease caused by biallelic mutations of the Ngly1 gene. Although epileptic seizure is one of the most severe symptoms in patients with...
NGLY1 deficiency is a genetic disease caused by biallelic mutations of the Ngly1 gene. Although epileptic seizure is one of the most severe symptoms in patients with NGLY1 deficiency, preclinical studies have not been conducted due to the lack of animal models for epileptic seizures in NGLY1 deficiency. Here, we observed the behaviors of male and female Ngly1 mice by video monitoring and found that these mice exhibit spontaneous seizure-like behaviors. Gene expression analyses and enzyme immunoassay revealed significant decreases in oxytocin, a well-known neuropeptide, in the hypothalamus of Ngly1 mice. Seizure-like behaviors in Ngly1 mice were transiently suppressed by a single intranasal administration of oxytocin. These findings suggest the therapeutic potential of oxytocin for epileptic seizure in patients with NGLY1 deficiency and contribute to the clarification of the disease mechanism.
Topics: Animals; Female; Male; Mice; Administration, Intranasal; Behavior, Animal; Disease Models, Animal; Hypothalamus; Mice, Inbred C57BL; Mice, Knockout; Oxytocin; Seizures; Congenital Disorders of Glycosylation; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
PubMed: 38649481
DOI: 10.1038/s42003-024-06131-7 -
Biochemistry. Biokhimiia Mar 2024Some tricyclic antidepressants (TCAs), including amitriptyline (ATL), clomipramine (CLO), and desipramine (DES), are known to be effective for management of neuropathic...
Some tricyclic antidepressants (TCAs), including amitriptyline (ATL), clomipramine (CLO), and desipramine (DES), are known to be effective for management of neuropathic pain. It was previously determined that ATL, CLO, and DES are capable of voltage-dependent blocking of NMDA receptors of glutamate (NMDAR), which play a key role in pathogenesis of neuropathic pain. Despite the similar structure of ATL, CLO, and DES, efficacy of their interaction with NMDAR varies significantly. In the study presented here, we applied molecular modeling methods to investigate the mechanism of binding of ATL, CLO, and DES to NMDAR and to identify structural features of the drugs that determine their inhibitory activity against NMDAR. Molecular docking of the studied TCAs into the NMDAR channel was performed. Conformational behavior of the obtained complexes in the lipid bilayer was simulated by the method of molecular dynamics (MD). A single binding site (upper) for the tertiary amines ATL and CLO and two binding sites (upper and lower) for the secondary amine DES were identified inside the NMDAR channel. The upper and lower binding sites are located along the channel axis at different distances from the extracellular side of the plasma membrane. MD simulation revealed that the position of DES in the lower site is stabilized only in the presence of sodium cation inside the NMDAR channel. DES binds more strongly to NMDAR compared to ATL and CLO due to simultaneous interaction of two hydrogen atoms of its cationic group with the asparagine residues of the ion pore of the receptor. This feature may be responsible for the stronger side effects of DES. It has been hypothesized that ATL binds to NMDAR less efficiently compared to DES and CLO due to its lower conformational mobility. The identified features of the structure- and cation-dependent mechanism of interaction between TCAs and NMDAR will help in the further development of effective and safe analgesic therapy.
Topics: Receptors, N-Methyl-D-Aspartate; Antidepressive Agents, Tricyclic; Molecular Docking Simulation; Molecular Dynamics Simulation; Binding Sites; Amitriptyline; Humans; Clomipramine; Cations; Desipramine; Protein Binding
PubMed: 38648769
DOI: 10.1134/S0006297924030106 -
Physical Chemistry Chemical Physics :... May 2024Soluble -glycosyltransferase from (ApNGT) catalyzes the glycosylation of asparagine residues, and represents one of the most encouraging biocatalysts for -glycoprotein...
Soluble -glycosyltransferase from (ApNGT) catalyzes the glycosylation of asparagine residues, and represents one of the most encouraging biocatalysts for -glycoprotein production. Since the sugar tolerance of ApNGT is restricted to limited monosaccharides (, Glc, GlcN, Gal, Xyl, and Man), tremendous efforts are devoted to expanding the substrate scope of ApNGT enzyme engineering. However, rational design of novel NGT variants suffers from an elusive understanding of the substrate-binding process from a dynamic point of view. Here, by employing extensive all-atom molecular dynamics (MD) simulations integrated with a kinetic model, we reveal, at the atomic level, the complete donor-substrate binding process from the bulk solvent to the ApNGT active-site, and the key intermediate states of UDP-Glc during its loading dynamics. We are able to determine the critical transition event that limits the overall binding rate, which guides us to pinpoint the key ApNGT residues dictating the donor-substrate entry. The functional roles of several identified gating residues were evaluated through site-directed mutagenesis and enzymatic assays. Two single-point mutations, N471A and S496A, could profoundly enhance the catalytic activity of ApNGT. Our work provides deep mechanistic insights into the structural dynamics of the donor-substrate loading process for ApNGT, which sets a rational basis for design of novel NGT variants with desired substrate specificity.
Topics: Actinobacillus pleuropneumoniae; Kinetics; Molecular Dynamics Simulation; Substrate Specificity; Glycosyltransferases; Mutagenesis, Site-Directed; Catalytic Domain
PubMed: 38647259
DOI: 10.1039/d3cp06034a -
Molecular and Cellular Biology 2024Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical models of inflammation. However, the molecular mechanisms...
Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor β (IκBβ). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBβ oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBβ oxomer formation did not occur via an IκBβ asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBβ ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBβ from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBβ contributes to the hypoxia-dependent regulation of inflammation.
Topics: Humans; NF-kappa B; I-kappa B Proteins; Protein Binding; Cell Hypoxia; Oxygen; HEK293 Cells; Mixed Function Oxygenases; Transcription Factor RelA; Animals; Hypoxia; Repressor Proteins
PubMed: 38644795
DOI: 10.1080/10985549.2024.2338727 -
The Journal of Biological Chemistry Jun 2024Cellular zinc ions (Zn) are crucial for signal transduction in various cell types. The transient receptor potential (TRP) ankyrin 1 (TRPA1) channel, known for its... (Comparative Study)
Comparative Study
Cellular zinc ions (Zn) are crucial for signal transduction in various cell types. The transient receptor potential (TRP) ankyrin 1 (TRPA1) channel, known for its sensitivity to intracellular Zn ([Zn]), has been a subject of limited understanding regarding its molecular mechanism. Here, we used metal ion-affinity prediction, three-dimensional structural modeling, and mutagenesis, utilizing data from the Protein Data Bank and AlphaFold database, to elucidate the [Zn] binding domain (IZD) structure composed by specific AAs residues in human (hTRPA1) and chicken TRPA1 (gTRPA1). External Zn induced activation in hTRPA1, while not in gTRPA1. Moreover, external Zn elevated [Zn] specifically in hTRPA1. Notably, both hTRPA1 and gTRPA1 exhibited inherent sensitivity to [Zn], as evidenced by their activation upon internal Zn application. The critical AAs within IZDs, specifically histidine at 983/984, lysine at 711/717, tyrosine at 714/720, and glutamate at 987/988 in IZD1, and H983/H984, tryptophan at 710/716, E854/E855, and glutamine at 979/980 in IZD2, were identified in hTRPA1/gTRPA1. Furthermore, mutations, such as the substitution of arginine at 919 (R919) to H919, abrogated the response to external Zn in hTRPA1. Among single-nucleotide polymorphisms (SNPs) at Y714 and a triple SNP at R919 in hTRPA1, we revealed that the Zn responses were attenuated in mutants carrying the Y714 and R919 substitution to asparagine and proline, respectively. Overall, this study unveils the intrinsic sensitivity of hTRPA1 and gTRPA1 to [Zn] mediated through IZDs. Furthermore, our findings suggest that specific SNP mutations can alter the responsiveness of hTRPA1 to extracellular and intracellular Zn.
Topics: Zinc; Humans; TRPA1 Cation Channel; Chickens; Animals; HEK293 Cells; Protein Domains; Species Specificity
PubMed: 38642892
DOI: 10.1016/j.jbc.2024.107302 -
BMC Psychiatry Apr 2024Despite ongoing research, the underlying causes of schizophrenia remain unclear. Aspartate and asparagine, essential amino acids, have been linked to schizophrenia in...
BACKGROUND
Despite ongoing research, the underlying causes of schizophrenia remain unclear. Aspartate and asparagine, essential amino acids, have been linked to schizophrenia in recent studies, but their causal relationship is still unclear. This study used a bidirectional two-sample Mendelian randomization (MR) method to explore the causal relationship between aspartate and asparagine with schizophrenia.
METHODS
This study employed summary data from genome-wide association studies (GWAS) conducted on European populations to examine the correlation between aspartate and asparagine with schizophrenia. In order to investigate the causal effects of aspartate and asparagine on schizophrenia, this study conducted a two-sample bidirectional MR analysis using genetic factors as instrumental variables.
RESULTS
No causal relationship was found between aspartate and schizophrenia, with an odds ratio (OR) of 1.221 (95%CI: 0.483-3.088, P-value = 0.674). Reverse MR analysis also indicated that no causal effects were found between schizophrenia and aspartate, with an OR of 0.999 (95%CI: 0.987-1.010, P-value = 0.841). There is a negative causal relationship between asparagine and schizophrenia, with an OR of 0.485 (95%CI: 0.262-0.900, P-value = 0.020). Reverse MR analysis indicates that there is no causal effect between schizophrenia and asparagine, with an OR of 1.005(95%CI: 0.999-1.011, P-value = 0.132).
CONCLUSION
This study suggests that there may be a potential risk reduction for schizophrenia with increased levels of asparagine, while also indicating the absence of a causal link between elevated or diminished levels of asparagine in individuals diagnosed with schizophrenia. There is no potential causal relationship between aspartate and schizophrenia, whether prospective or reverse MR. However, it is important to note that these associations necessitate additional research for further validation.
Topics: Humans; Asparagine; Aspartic Acid; Schizophrenia; Genome-Wide Association Study; Mendelian Randomization Analysis; Prospective Studies
PubMed: 38641826
DOI: 10.1186/s12888-024-05765-5