Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant.

A metabolic map of the DNA damage response identifies PRDX1 in the control of nuclear ROS scavenging and aspartate availability.

While cellular metabolism impacts the DNA damage response, a systematic understanding of the metabolic requirements that are crucial for DNA damage repair has yet to be achieved. Here, we investigate the metabolic enzymes and processes that are essential for the resolution of DNA damage. By integrating functional genomics with chromatin proteomics and metabolomics, we provide a detailed description of the interplay between cellular metabolism and the DNA damage response. Further analysis identified that Peroxiredoxin 1, PRDX1, contributes to the DNA damage repair. During the DNA damage response, PRDX1 translocates to the nucleus where it reduces DNA damage-induced nuclear reactive oxygen species. Moreover, PRDX1 loss lowers aspartate availability, which is required for the DNA damage-induced upregulation of de novo nucleotide synthesis. In the absence of PRDX1, cells accumulate replication stress and DNA damage, leading to proliferation defects that are exacerbated in the presence of etoposide, thus revealing a role for PRDX1 as a DNA damage surveillance factor.

Asparagine reduces the risk of schizophrenia: a bidirectional two-sample mendelian randomization study of aspartate, asparagine and schizophrenia.

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.

A short amphipathic alpha helix in scavenger receptor BI facilitates bidirectional HDL-cholesterol transport.

During reverse cholesterol transport, high-density lipoprotein (HDL) carries excess cholesterol from peripheral cells to the liver for excretion in bile. The first and last steps of this pathway involve the HDL receptor, scavenger receptor BI (SR-BI). While the mechanism of SR-BI-mediated cholesterol transport has not yet been established, it has long been suspected that cholesterol traverses through a hydrophobic tunnel in SR-BI's extracellular domain. Confirmation of a hydrophobic tunnel is hindered by the lack of a full-length SR-BI structure. Part of SR-BI's structure has been resolved, encompassing residues 405 to 475, which includes the C-terminal transmembrane domain and its adjacent extracellular region. Within the extracellular segment is an amphipathic helix (residues 427-436, referred to as AH(427-436)) that showed increased protection from solvent in NMR-based studies. Homology models predict that hydrophobic residues in AH(427-436) line a core cavity in SR-BI's extracellular region that may facilitate cholesterol transport. Therefore, we hypothesized that hydrophobic residues in AH(427-436) are required for HDL cholesterol transport. Here, we tested this hypothesis by mutating individual residues along AH(427-436) to a charged residue (aspartic acid), transiently transfecting COS-7 cells with plasmids encoding wild-type and mutant SR-BI, and performing functional analyses. We found that mutating hydrophobic, but not hydrophilic, residues in AH(427-436) impaired SR-BI bidirectional cholesterol transport. Mutating phenylalanine-430 was particularly detrimental to SR-BI's functions, suggesting that this residue may facilitate important interactions for cholesterol delivery within the hydrophobic tunnel. Our results support the hypothesis that a hydrophobic tunnel within SR-BI mediates cholesterol transport.

Optical Control of Protein Functions via Genetically Encoded Photocaged Aspartic Acids.

Site-specific protein decaging by light has become an effective approach for in situ manipulation of protein activities in a gain-of-function fashion. Although successful decaging of amino acid side chains of Lys, Tyr, Cys, and Glu has been demonstrated, this strategy has not been extended to aspartic acid (Asp), an essential amino acid residue with a range of protein functions and protein-protein interactions. We herein reported a genetically encoded photocaged Asp and applied it to the photocontrolled manipulation of a panel of proteins including firefly luciferase, kinases (e.g., BRAF), and GTPase (e.g., KRAS) as well as mimicking the in situ phosphorylation event on kinases. As a new member of the increasingly expanded amino acid-decaging toolbox, photocaged Asp may find broad applications for gain-of-function study of diverse proteins as well as biological processes in living cells.

Isolation and functional verification of an aspartate aminotransferase gene from Neoporphyra haitanensis.

BACKGROUND: Neoporphyra haitanensis is a commercial laver species in China. Aspartic acid is an important flavor amino acid, and aspartate aminotransferase (AAT) is a crucial enzyme in its biosynthesis. In this study, we cloned one AAT gene (NhAAT) from the red alga N. haitanensis and investigated its sequence structure, transcriptional expression and enzymatic characteristics. The purpose of our research is to obtain a functional AAT responsible for the biosynthesis of aspartic acid from red seaweeds, which has the potential to influence the flavor of N. haitanensis. RESULTS: Sequence analysis showed that NhAAT contains a conserved domain of Aminotran_1_2, which belongs to the transaminase superfamily. The secondary structure of NhAAT is dominated by α-helix. The results of enzymatic characterization illustrated that the NhAAT has highest catalytic activity at 45 °C and pH 7.5 in both forward and reverse reactions. The calculated Km values of NhAAT was 5.67 and 6.16 mM for L-glutamic acid and L-aspartic acid, respectively. Quantitative analysis showed that the NhAAT expression of N. haitanensis collected in late harvest (Dec) was 4.5 times that of N. haitanensis collected in early harvest (Oct), while the aspartic acid content of N. haitanensis collected in late harvest (Dec) was 1.2 times that of N. haitanensis collected in early harvest (Oct). CONCLUSION: The results of enzyme kinetics indicated that NhAAT prefers to catalyze the reaction in the direction of aspartic acid production. Moreover, the trend of NhAAT expression level was consistent with that of aspartic acid content in N. haitanensis in different harvest periods. Our research is helpful to understand the accumulation and regulation of amino acids in N. haitanensis in different habitats and the taste difference of N. haitanensis in different harvest periods.

FN1 mediated activation of aspartate metabolism promotes the progression of triple-negative and luminal a breast cancer.

BACKGROUND: Breast cancer (BC) is regarded as one of the most common cancers diagnosed among the female population and has an extremely high mortality rate. It is known that Fibronectin 1 (FN1) drives the occurrence and development of a variety of cancers through metabolic reprogramming. Aspartic acid is considered to be an important substrate for nucleotide synthesis. However, the regulatory mechanism between FN1 and aspartate metabolism is currently unclear. METHODS: We used RNA sequencing (RNA seq) and liquid chromatography-mass spectrometry to analyze the tumor tissues and paracancerous tissues of patients. MCF7 and MDA-MB-231 cells were used to explore the effects of FN1-regulated aspartic acid metabolism on cell survival, invasion, migration and tumor growth. We used PCR, Western blot, immunocytochemistry and immunofluorescence techniques to study it. RESULTS: We found that FN1 was highly expressed in tumor tissues, especially in Lumina A and TNBC subtypes, and was associated with poor prognosis. In vivo and in vitro experiments showed that silencing FN1 inhibits the activation of the YAP1/Hippo pathway by enhancing YAP1 phosphorylation, down-regulates SLC1A3-mediated aspartate uptake and utilization by tumor cells, inhibits BC cell proliferation, invasion and migration, and promotes apoptosis. In addition, inhibition of FN1 combined with the YAP1 inhibitor or SLC1A3 inhibitor can effectively inhibit tumor growth, of which inhibition of FN1 combined with the YAP1 inhibitor is more effective. CONCLUSION: Targeting the "FN1/YAP1/SLC1A3/Aspartate metabolism" regulatory axis provides a new target for BC diagnosis and treatment. This study also revealed that intratumoral metabolic heterogeneity plays an important role in the progression of different subtypes of breast cancer.

Wild soybean salt tolerance metabolic model: Assessment of storage protein mobilization in cotyledons and C/N balance in the hypocotyl/root axis.

Salt stress has become one of the main factors limiting crop yield in recent years. The post-germinative growth is most sensitive to salt stress in soybean. In this study, cultivated and wild soybeans were used for an integrated metabonomics and transcriptomics analysis to determine whether wild soybean can resist salt stress by maintaining the mobilization of stored substances in cotyledons and the balance of carbon and nitrogen in the hypocotyl/root axis (HRA). Compared with wild soybean, the growth of cultivated soybean was significantly inhibited during the post-germinative growth period under salt stress. Integrating analysis found that the breakdown products of proteins, such as glutamate, glutamic acid, aspartic acid, and asparagine, increased significantly in wild soybean cotyledons. Asparagine synthase and fumarate hydratase genes and genes encoding HSP20 family proteins were specifically upregulated. In wild soybean HRA, levels of glutamic acid, aspartic acid, asparagine, citric acid, and succinic acid increased significantly, and the glutamate decarboxylase gene and the gene encoding carbonic anhydrase in nitrogen metabolism were significantly upregulated. The metabolic model indicated that wild soybean enhanced the decomposition of stored proteins and the transport of amino acids to the HRA in cotyledons and the GABA shunt to maintain carbon and nitrogen balance in the HRA to resist salt stress. This study provided a theoretical basis for cultivating salt-tolerant soybean varieties and opened opportunities for the development of sustainable agricultural practices.

Long noncoding RNA LINC01234 promotes hepatocellular carcinoma progression through orchestrating aspartate metabolic reprogramming.

Amino acids metabolism, especially aspartate metabolism, is often altered in human cancers including hepatocellular carcinoma (HCC) and this metabolic remodeling is required for supporting cancer cell malignant activities. Argininosuccinate synthase 1 (ASS1), as a crucial rate-limiting enzyme in aspartate metabolism, participates in repressing tumor progression. However, the roles of long noncoding RNAs (lncRNAs) in aspartate metabolism remodeling and the underlying mechanisms remain unclear. Here, we screen LINC01234 as an aspartate metabolism-related lncRNA in HCC. Clinically, LINC01234 was highly expressed in HCC, and a high LINC01234 expression level was correlated with a poor prognosis of patients with HCC. LINC01234 promoted cell proliferation, migration, and drug resistance by orchestrating aspartate metabolic reprogramming in HCC cells. Mechanistically, LINC01234 downregulated the expression of ASS1, leading to am increased aspartate level and activation of the mammalian target of rapamycin pathway. LINC01234 bound to the promoter of ASS1 and inhibited transcriptional activation of ASS1 by transcriptional factors, including p53. Finally, inhibiting LINC01234 dramatically impaired tumor growth in nude mice and sensitized HCC cells to sorafenib. These findings demonstrate that LINC01234 promotes HCC progression by modulating aspartate metabolic reprogramming and might be a prognostic or therapeutic target for HCC.

Alternative proton-binding site and long-distance coupling in Escherichia coli sodium-proton antiporter NhaA.

Escherichia coli NhaA is a prototypical sodium-proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.

Specific Mutations near the Amyloid Precursor Protein Cleavage Site Increase γ-Secretase Sensitivity and Modulate Amyloid-ß Production.

Amyloid-ß peptides (Aßs) are produced via cleavage of the transmembrane region of the amyloid precursor protein (APP) by γ-secretase and are responsible for Alzheimer's disease. Familial Alzheimer's disease (FAD) is associated with APP mutations that disrupt the cleavage reaction and increase the production of neurotoxic Aßs, i.e., Aß42 and Aß43. Study of the mutations that activate and restore the cleavage of FAD mutants is necessary to understand the mechanism of Aß production. In this study, using a yeast reconstruction system, we revealed that one of the APP FAD mutations, T714I, severely reduced the cleavage, and identified secondary APP mutations that restored the cleavage of APP T714I. Some mutants were able to modulate Aß production by changing the proportions of Aß species when introduced into mammalian cells. Secondary mutations include proline and aspartate residues; proline mutations are thought to act through helical structural destabilization, while aspartate mutations are thought to promote interactions in the substrate binding pocket. Our results elucidate the APP cleavage mechanism and could facilitate drug discovery.

Effect of ACGT motif in spatiotemporal regulation of AtAVT6D, which improves tolerance to osmotic stress and nitrogen-starvation.

KEY MESSAGE: Plasma membrane-localized AtAVT6D importing aspartic acid can be targeted to develop plants with enhanced osmotic and nitrogen-starvation tolerance. AtAVT6D promoter can be exploited as a stress-inducible promoter for genetic improvements to raise stress-resilient crops. The AtAVT6 family of amino acid transporters in Arabidopsis thaliana has been predicted to export amino acids like aspartate and glutamate. However, the functional characterization of these amino acid transporters in plants remains unexplored. The present study investigates the expression patterns of AtAVT6 genes in different tissues and under various abiotic stress conditions using quantitative Real-time PCR. The expression analysis demonstrated that the member AtAVT6D was significantly induced in response to phytohormone ABA and stresses like osmotic and drought. The tissue-specific expression analysis showed that AtAVT6D was strongly expressed in the siliques. Taking together these results, we can speculate that AtAVT6D might play a vital role in silique development and abiotic stress tolerance. Further, subcellular localization study showed AtAVT6D was localized to the plasma membrane. The heterologous expression of AtAVT6D in yeast cells conferred significant tolerance to nitrogen-deficient and osmotic stress conditions. The Xenopus oocyte studies revealed that AtAVT6D is involved in the uptake of Aspartic acid. While overexpression of AtAVT6D resulted in smaller siliques in Arabidopsis thaliana. Additionally, transient expression studies were performed with the full-length AtAVT6D promoter and its deletion constructs to study the effect of ACGT-N24-ACGT motifs on the reporter gene expression in response to abiotic stresses and ABA treatment. The fluorometric GUS analyses revealed that the promoter deletion construct-2 (Pro.C2) possessing a single copy of ACGT-N24-ACGT motif directed the strongest GUS expression under all the abiotic conditions tested. These results suggest that Pro.C2 can be used as a stress-inducible promoter to drive a significant transgene expression.

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

Genetic disruption of N-RasG12D palmitoylation perturbs hematopoiesis and prevents myeloid transformation in mice.

Oncogenic RAS mutations pose substantial challenges for rational drug discovery. Sequence variations within the hypervariable region of Ras isoforms underlie differential posttranslational modification and subcellular trafficking, potentially resulting in selective vulnerabilities. Specifically, inhibiting the palmitoylation/depalmitoylation cycle is an appealing strategy for treating NRAS mutant cancers, particularly as normal tissues would retain K-Ras4b function for physiologic signaling. The role of endogenous N-RasG12D palmitoylation in signal transduction, hematopoietic differentiation, and myeloid transformation is unknown, and addressing these key questions will inform efforts to develop mechanism-based therapies. To evaluate the palmitoylation/depalmitoylation cycle as a candidate drug target in an in vivo disease-relevant model system, we introduced a C181S mutation into a conditional NrasG12D "knock-in" allele. The C181S second-site amino acid substitution abrogated myeloid transformation by NrasG12D, which was associated with mislocalization of the nonpalmitoylated N-Ras mutant protein, reduced Raf/MEK/ERK signaling, and alterations in hematopoietic stem and progenitor populations. Furthermore, hematologic malignancies arising in NrasG12D/G12D,C181S compound heterozygous mice invariably acquired revertant mutations that restored cysteine 181. Together, these studies validate the palmitoylation cycle as a promising therapeutic target in NRAS mutant cancers.

Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase.

The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B. Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function and may facilitate the development of AIV-resistant chickens via precise modification of residues within ANP32A.

A Polyplex in a Shell: The Effect of Poly(aspartic acid)-Mediated Calcium Carbonate Mineralization on Polyplexes Properties and Transfection Efficiency.

Mineralization by exposure of organic templates to supersaturated solutions is used by many living organisms to generate specialized materials to perform structural or protective functions. Similarly, it was suggested that improved robustness acquired through mineralization under natural conditions could be an important factor for virus survival outside of a host for better transfection of cells. Here, inspired by this fact, we developed a nonviral tricomponent polyplex system for gene delivery capable of undergoing mineralization. First, we fabricated anionic polyplexes carrying pDNA by self-assembly with a lipid-modified cationic polymer and coating by poly(aspartic acid). Then, we submitted the polyplexes to a two-step mineralization reaction to precipitate CaCO3 under various supersaturations. We carried out detailed morphological studies of the mineralized polyplexes and identified which parameters of the fabrication process were influential on transfection efficiency. We found that mineralization with CaCO3 is efficient in promoting transfection efficiency as long as a certain Ca2+/CO32- lower limit ratio is respected. However, calcium incubation can also be used to achieve similar effects at higher concentrations depending on polyplex composition, probably due to the formation of physical cross-links by calcium binding to poly(aspartic acid). We proposed that the improved robustness and transfection efficiency provided by means of mineralization can be used to expand the possible applications of polyplexes in gene therapy.

The pncA gene mutations of Mycobacterium tuberculosis in multidrug-resistant tuberculosis.

The pncA gene encodes pyrazinamidase enzyme which converts drug pyrazinamide to active form pyrazinoic acid, but mutations in this gene can prevent enzyme activity which leads to pyrazinamide resistance. The cross-sectional study was carried out during 2016-2017 for 12 months. The purpose of the study was to detect mutation at codon 12 and codon 85 in the pncA gene in local multidrug-resistant tuberculosis (MDR-TB) patients by developing a simple molecular test so that disease could be detected timely in the local population. DNA extracted from sputum-cultured samples from MDR-TB patients and subjected to semi-multiplex allele-specific PCR by using self-designed primers against the pncA gene. Among 75 samples, 53 samples were subjected to molecular analysis based on purified DNA quantity and quality. The primers produced 250 and 480 bp fragments, indicating the mutations at codon 12 (aspartate to alanine) and codon 85 (leucine to proline) respectively. MDR-TB was more common in the age group 21-40 years. Fifty-seven percent of samples (n = 30) were found positive for pncA mutations, whereas 43% of samples (n = 23) showed negative results. Thirteen percent of samples (n = 4) had mutations at codon 12 in which aspartate was converted to alanine, and they produced an amplified product of 480 bp. Eighty-seven percent of samples (n = 26) had mutations at codon 85 in which leucine was converted to proline and amplified product size was 250 bp. The mutations were simple nucleotide substitutions. The prevalence of mutations in which leucine was substituted by proline was higher than the mutations in which aspartate was substituted by alanine. A high prevalence of substitution mutation (CTG → CCG; leucine to proline) was detected in MDR-TB cases. Earlier detection of MDR-TB via an effective molecular diagnostic method can control the MDR tuberculosis spread in the population.

Characterization of tolloid-mediated cleavage of the GDF8 procomplex.

Growth differentiation factor 8 (GDF8), a.k.a. myostatin, is a member of the larger TGFß superfamily of signaling ligands. GDF8 has been well characterized as a negative regulator of muscle mass. After synthesis, GDF8 is held latent by a noncovalent complex between the N-terminal prodomain and the signaling ligand. Activation of latent GDF8 requires proteolytic cleavage of the prodomain at residue D99 by a member of the tolloid family of metalloproteases. While tolloid proteases cleave multiple substrates, they lack a conserved consensus sequence. Here, we investigate the tolloid cleavage site of the GDF8 prodomain to determine what residues contribute to tolloid recognition and subsequent proteolysis. Using sequential alanine mutations, we identified several residues adjacent to the scissile bond, including Y94, that when mutated, abolish tolloid-mediated activation of latent GDF8. Using the astacin domain of Tll1 (Tolloid Like 1) we determined that prodomain mutants were more resistant to proteolysis. Purified latent complexes harboring the prodomain mutations, D92A and Y94A, impeded activation by tolloid but could be fully activated under acidic conditions. Finally, we show that co-expression of GDF8 WT with prodomain mutants that were tolloid resistant, suppressed GDF8 activity. Taken together our data demonstrate that residues towards the N-terminus of the scissile bond are important for tolloid-mediated activation of GDF8 and that the tolloid-resistant version of the GDF8 prodomain can function dominant negative to WT GDF8.

A Cofactor-Based Mechanism for the Origin of the Genetic Code.

The origin of the genetic code is probably the central problem of the studies on the origin of life. The key question to answer is the molecular mechanism that allows the association of the amino acids with their triplet codons. We proposed that the codon-anticodon duplex located in the acceptor stem of primitive tRNAs would facilitate the chemical reactions required to synthesize cognate amino acids from simple amino acids (glycine, valine, and aspartic acid) linked to the 3' acceptor end. In our view, various nucleotide-A-derived cofactors (with reactive chemical groups) may be attached to the codon-anticodon duplex, which allows group-transferring reactions from cofactors to simple amino acids, thereby producing the final amino acid. The nucleotide-A-derived cofactors could be incorporated into the RNA duplex (helix) by docking Adenosine (cofactor) into the minor groove via an interaction similar to the A-minor motif, forming a base triple between Adenosine and one complementary base pair of the duplex. Furthermore, we propose that this codon-anticodon duplex could initially catalyze a self-aminoacylation reaction with a simple amino acid. Therefore, the sequence of bases in the codon-anticodon duplex would determine the reactions that occurred during the formation of new amino acids for selective binding of nucleotide-A-derived cofactors.

Toxicity effects of chlorantraniliprole in zebrafish (Danio rerio) involving in liver function and metabolic phenotype.

Chlorantraniliprole (CAP), a representative bisamide insecticide, is widely used in rice fields around the world, posing potential toxicity risks to aquatic organisms. In this study, we examined the effects of exposure to CAP on growth and metabolic phenotype of zebrafish (Danio rerio) and oxidative stress and apoptosis in the liver of zebrafish (Danio rerio). First, we identified that CAP had a low bioaccumulation in zebrafish. Subsequently, growth phenotype analysis revealed that CAP could significantly increase liver weight and liver index in zebrafish. In addition, we found that CAP exposure could cause significant changes in indicators of oxidative stress, resulting in a significant increase in the content of malondialdehyde (MDA), causing oxidative stress in the liver of zebrafish. Meanwhile, the expression levels of apoptosis-related genes were also significantly changed and apoptosis was promoted in the liver of zebrafish with CAP exposure. Importantly, the results of metabolomics analysis shown that CAP exposure could significantly disrupt the metabolic phenotype of zebrafish, interfering with multiple metabolic pathways, mainly including valine, leucine and isoleucine biosynthesis and degradation, alanine, aspartate and glutamate metabolism and d-glutamine and D-glutamate metabolism. Last but not least, correlation analysis identified strong links between changes in liver function involving oxidative stress and apoptosis and changes in metabolic phenotype of zebrafish following CAP exposure. In brief, these results indicate that potential environmental risks of CAP to aquatic organisms should receive more attention.