Changes In Amino Acid Sequence Research Articles

Primary amino acid sequences of decapod (Na+, K+)-ATPase provide evolutionary insights into osmoregulatory mechanisms

Decapod Crustacea exhibit a marine origin, but many taxa have occupied environments ranging from brackish to fresh water and terrestrial habitats, overcoming their inherent osmotic challenges. Osmotic and ionic regulation is achieved by the gill epithelia, driven by two active ATP-hydrolyzing ion transporters, the basal (Na+, K+)-ATPase and the apical V(H+)-ATPase. The kinetic characteristic of gill (Na+, K+)-ATPase and the mRNA expression of its alpha subunit have been widely studied in various decapod species under different salinity challenges. However, the evolution of the primary structure has not been explored, especially considering the functional modifications associated with decapod phylogeny. Here, we proposed a model for the topology of the decapod alpha subunit, identifying the sites and motifs involved in its function and regulation, as well as the patterns of its evolution assuming a decapod phylogeny. We also examined both the amino acid substitutions and their functional implications within the context of biochemical and physiological adaptation. The α-subunit of decapod crustaceans shows greater conservation (~94% identity) compared to the β-subunit (~40%). While the binding sites for ATP and modulators are conserved in the decapod enzyme, the residues involved in the α-β interaction are only partially conserved. In the phylogenetic context of the complete sequence of (Na+, K+)-ATPase α-subunit, most substitutions appear to be characteristic of the entire group, with specific changes for different subgroups, especially among brachyuran crabs. Interestingly, there was no consistent separation of α-subunit partial sequences related to habitat, suggesting that the convergent evolution for freshwater or terrestrial modes of life is not correlated with similar changes in the enzyme's primary amino acid sequence.

Abstract 2767: Exploring ADAR1 dependencies in breast cancer brain-metastases

Abstract Background: Adenosine deaminase acting on double-stranded RNA (ADAR1) is a recognized driver of epitranscriptomic (post-transcriptional) remodeling and tumor progression. Changes in amino acid sequence, mRNA splicing, and/or polyadenylation sites are only some of the adenosine-to-inosine (A-to-I) RNA editing effects. Under physiological conditions, ADAR1 activity prevents autoinflammation by inhibiting the accumulation of endogenous immunostimulatory double-stranded RNA and by suppressing the Z-RNA sensor ZBP1. Such mechanisms are exploited by cancer cells to desensitize tumor response to therapies, therefore augmenting aggressiveness. Approximately 10 to 20% of patients diagnosed with breast cancer (BC) will develop metastatic disease, which remains the leading cause of death in individuals with BC. We have hypothesized that ADAR1 and its interferon-inducible p150 isoform, play a pivotal role in promoting BC metastases since high levels of ADAR1 are associated with a worse prognosis in patients with high grade BC, especially in the triple-negative BC subgroup (TNBC), which is the most likely to develop brain metastases. Methods: We established a humanized mouse model of breast cancer brain metastases (BC-BrM) using neonatal intracerebroventricular inoculation of MDA-MD-231 TNBC cells and primary patient derived metastatic cells in Rag2−/−γc−/− mice. Before implantation, MDA-MD-231 cells were transduced with a nanoluc-GFP reporter for tracing ADAR1 enzymatic activity in the brain metastatic niche. Lentiviral shRNA knockdown vectors targeting ADAR1, combined with over-expression vectors of full-length ADAR1 or the p150 deleted isoform were adopted to detect editing changes in the metastatic model through RNA-sequencing of BC-BrM. Results: BrM derived from MDA-MD-231 implants display upregulation of ADAR1 p150 and activation of the WNT/β-catenin pathway compared to the non-implanted cell lines. Upon shRNA knockdown of ADAR1, decreased expression of BC stem cell markers was detected via FACS in MDA-MD-231 ADAR1 knocked-down versus control BC-BrM isolated cells. We are now exploring molecular pathways affected by ADAR1 loss/overexpression in establishing BrM. Future Directions: We have developed a biorepository of primary patient-derived metastatic breast cancer samples and have begun the process of tracking metastatic kinetics and ADAR1 activation with the ADAR1 reporter in an orthotopic mouse model. Overall, this study will provide new insights into the molecular role of ADAR1 deaminase activity in establishing and maintaining metastatic niches. Citation Format: Teresa Sposito, Emma E. Klacking, Antonio W. Ruiz, Jessica Pham, Catriona Jamieson. Exploring ADAR1 dependencies in breast cancer brain-metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2767.

Identifying and Characterizing Candidate Genes Contributing to a Grain Yield QTL in Wheat.

The current study focuses on identifying the candidate genes of a grain yield QTL from a double haploid population, Westonia × Kauz. The QTL region spans 20 Mbp on the IWGSC whole-genome sequence flank with 90K SNP markers. The IWGSC gene annotation revealed 16 high-confidence genes and 41 low-confidence genes. Bioinformatic approaches, including functional gene annotation, ontology investigation, pathway exploration, and gene network study using publicly available gene expression data, enabled the short-listing of four genes for further confirmation. Complete sequencing of those four genes demonstrated that only two genes are polymorphic between the parental cultivars, which are the ferredoxin-like protein gene and the tetratricopeptide-repeat (TPR) protein gene. The two genes were selected for downstream investigation. Two SNP variations were observed in the exon for both genes, with one SNP resulting in changes in amino acid sequence. qPCR-based gene expression showed that both genes were highly expressed in the high-yielding double haploid lines along with the parental cultivar Westonia. In contrast, their expression was significantly lower in the low-yielding lines in the other parent. It can be concluded that these two genes are the contributing genes to the grain yield QTL.

Identification of new nucleotide sequences of the Glu-B1-1 gene encoding x-type glutenins in bread wheat.

Studies of the genetic base and polymorphism of bread wheat cultivars aimed at identifying alleles of genes associated with high baking and other economically valuable traits seem to be relevant, since bread wheat, along with all representatives of the Triticeae tribe, has a huge genetic potential for creating cultivars with high technological and rheological properties of grain flour. The aim of this study was sequencing and analysis of the nucleotide sequences of the Glu-B1-1 gene, and analysis of the predicted amino acid sequences of its protein product in three cultivars of bread wheat. Thus, in the course of genotyping cultivars and lines of bread wheat for the Glu-B1-1 gene, in the cultivars 'Avesta', 'Leningradka krupnozernaya' and line C-75094, previously undescribed changes in the size of amplifiable regions of the Glu-B1-1 gene for high-molecular weight glutenins were found. Comparative analysis of the nucleotide sequences of these genes with known sequences showed the presence of two deletions in 'Avesta' and C-75094 and the presence of seven single-nucleotide substitutions in 'Leningradka krupnozernaya'. Alignment of the predicted Glu-B1 amino acid sequences of the studied accessions and the standard cultivar carrying the Glu-B1-a allele showed that deletions in the amino acid sequences of 'Avesta' and C-75094 accessions are localized in the central domain of the protein and affect the amount of tri-, hexa-, and nonapeptides, and in 'Leningradka krupnozernaya', a decrease in GQQ and PGQGQQ by one unit was revealed. In addition, substitutions of five amino acids were found in 'Leningradka krupnozernaya'. Thus, we have found previously undescribed deletions and substitutions in the nucleotide sequences of the Glu-B1-1 gene for high-molecular-weight glutenins, which lead to changes in amino acid sequences in functionally important regions, namely, in the central domains of protein molecules. The identified mutations can be used for genotyping bread wheat cultivars.

Spontaneous mutational patterns and novel mutations for bedaquiline and clofazimine resistance in Mycobacterium tuberculosis.

The 2022 World Health Organization guidelines recommend use of two core anti-tuberculosis (TB) drugs, bedaquiline (BDQ) and clofazimine (CFZ), for treatment of drug-resistant (DR)-TB. However, several mutated Mycobacterium tuberculosis (MTB) genes, conferring BDQ and CFZ resistance, have been reported that predominantly arose from sporadic mutations that have not been comprehensively characterized. Herein, MTB clinical isolates collected from drug-susceptible (DS)-, multidrug-resistant (MDR)-, and extensively drug-resistant (XDR)-TB patients were cultured in vitro with BDQ or CFZ to generate progeny strains with resistance to these drugs. Progeny strains exposed to CFZ exhibited increased CFZ minimum inhibitory concentrations (MICs) that exceeded MIC increases of BDQ-exposed progeny strains. Notably, mmpR and pepQ mutations accounted for 83% and 17% of BDQ-induced spontaneous gene mutations, respectively, and 86% and 14% of CFZ-induced spontaneous gene mutations, respectively. Analyses of predicted mutation-induced changes in amino acid sequences and structures of MmpR and PepQ mutants revealed several point mutations affected sequence conversation and functionality as an underlying mechanism for observed acquired BDQ/CFZ resistance. Moreover, our results revealed differences in patterns of BDQ- and CFZ-induced acquired spontaneous mutations that may enhance our understanding of MTB BDQ/CFZ-resistance mechanisms. IMPORTANCE This study of MTB drug resistance mechanisms revealed patterns of spontaneous MTB mutations associated with acquired BDQ and CFZ resistance that arose after clinical MTB isolates were cultured in vitro with BDQ or CFZ. Results of protein sequence and structural analyses provided insights into potential mechanisms underlying associations between MTB gene mutations and DR phenotypes. Taken together, these results revealed differences in acquired BDQ and CFZ resistance mechanisms as a new perspective that may enhance our understanding of BDQ/CFZ resistance mechanisms and facilitate the development of new methods for detecting MTB drug resistance genes.

<i>In silico</i> analysis of genomes of Bacillus anthracis strains belonging to major genetic lineages

Introduction. The global phylogenetic population structure of Bacillus anthracis is represented by major genetic lineages (A, B and C) with nonuniform distribution of isolates, which still cannot be explained. Identification of characteristics of genomes of strains from three lineages, which can affect their spread, is of high importance.
 The aim of the study is to explore genomic characteristics of different genetic lineages, which may have an effect on their distribution, by using the in silico analysis of a representative subset of B. anthracis strains.
 Materials and methods. The whole-genome sequences of 49 B. anthracis strains and Bacillus cereus biovar anthracis CI strain were studied. The in silico analysis was performed to identify polymorphisms using BLASTn, MEGA X, Tandem Repeat Finder, Parsnp the Harvest Suite software.
 Results. The genome variability depended on single nucleotide polymorphisms, single-nucleotide repeats, number of tandem repeats, substitutions and indels. In strains from lineages B and C, they outnumbered 1.613.4 times and in the B. cereus biovar anthracis strain 5150 times those in B. anthracis strains from lineage A. Significant substitutions in housekeeping genes and pathogenicity factor genes caused changes in amino acid sequences in proteins significantly more frequently in B. anthracis strains from major lineages B and C.
 Based on the molecular typing and a multi-virulence-locus sequence typing analysis (MVLST) with a discrimination index of 0.9633, strains were classified into three major genetic lineages including groups different from the canonical group.
 Conclusion. The distinctive feature of B. anthracis genomes is that they have a larger number of significant nucleotide substitutions in pathogenicity factor genes and housekeeping genes of strains belonging to major lineages B and C compared to lineage A. Changes in proteins encoded by them can cause differences in ecological adaptation and in prevalence, which are higher in strains of lineage A. MVLST having a high discriminating capacity can be used as an additional method to B. anthracis molecular typing.

Plant Antimicrobial Peptides: Insights into Structure-Function Relationships for Practical Applications

Antimicrobial peptides (AMPs) are short polypeptide molecules produced by multicellular organisms that are involved in host defense and microbiome preservation. In recent years, AMPs have attracted attention as novel drug candidates. However, their successful use requires detailed knowledge of the mode of action and identification of the determinants of biological activity. In this review, we focused on structure-function relationships in the thionins, α-hairpinins, hevein-like peptides, and the unique Ib-AMP peptides isolated from Impatiens balsamina. We summarized the available data on the amino acid sequences and 3D structure of peptides, their biosynthesis, and their biological activity. Special attention was paid to the determination of residues that play a key role in the activity and the identification of the minimal active cores. We have shown that even subtle changes in amino acid sequences can affect the biological activity of AMPs, which opens up the possibility of creating molecules with improved properties, better therapeutic efficacy, and cheaper large-scale production.

Experimental reconstruction of membrane transporter evolution.

Cellular homeostasis relies on solutes crossing cell membranes against their concentration gradients. Membrane transporters catalyze this energetically unfavorable movement using energy from the downhill movement of ions. Often, related transporters couple to either Na+ or H+ gradients; it can be difficult to glean the mechanistic reasons between such differences because the ability to use ions with such different physicochemical properties might require many changes in the transporter. Since evolutionary adaptations to environmental constraints such as salt concentrations and pH likely gave rise to the switch between sodium- and proton-motive forces, we hypothesized that phylogenetic reconstruction of ion-coupling may be a method to study such complex changes. We used phylogenetic analysis to reconstruct the evolution of prokaryotic glutamate transporters and deduce stepwise amino acid sequence changes resulting in the emergence of H+-coupled transporters from Na+-coupled ancestors. The analysis suggests that in addition to changing protonatable residues, introducing H+-dependence increases the hydrophobicity of previously polar regions proximal to the substrate and Na+ binding sites. To contextualize these changes, we used ancestral sequence reconstruction (ASR) to infer transporter sequences before and after the emergence of H+-coupling. We expressed and purified the inferred ancestral transporters, enabling their biochemical characterization and high-resolution cryo-EM structures. Surprisingly, the ancestral transporter before the emergence of H+-coupling shows Na+-independent substrate binding even though its Na+-binding pockets are structurally near-identical to Na+-coupled homologs. The emergence of H+-coupling preserves the protein architecture while subtly restructuring these critical regions. Our results suggest that changes in local structure and complex global allosteric properties underlie the diversification of the ion-motive forces used within the glutamate transporter family. The approach we developed might be broadly applicable to dissecting complex allosteric properties of membrane proteins.

A Comprehensive Study on Antibiotic Resistance among Coagulase-Negative Staphylococci (CoNS) Strains Isolated from Ready-to-Eat Food Served in Bars and Restaurants.

The present study aimed to characterize and assess the diversity of CoNS strains as potential vectors for the spread of resistance to antimicrobial agents from RTE foods served in bars and restaurants. Eighty-five CoNS strains, obtained from 198 RTE food samples, were investigated. Sixty-seven CoNS isolates (78.8%) were resistant to at least one antibiotic tested, and 37 (43.5%) were multidrug resistant (MDR-CoNS). Moreover, CoNS strains contained genes conferring resistance to antibiotics critically important in medicine, i.e., β-lactams [mecA (29.4%); blaZ (84.7%)], aminoglycosides [aac(6')-Ie-aph(2″)-Ia (45.9%); aph(2″)-Ic (3.5%)], macrolides, lincosamides and streptogramin B-MLSB [msrA/B (68.2%); ermB (40%) and mphC (4.7%)], tetracyclines [tetK (31.8%); tetM (16.5%) and/or tetL (2.35%)]. We also found the fusB/C/D genes responsible for the acquired low-level fusidic acid resistance (17.6%) and streptogramin resistance determinant vgaA in 30.6% of isolates. In three linezolid resistant strains (2 S. epidermidis and 1 S. warneri), mutation was detected, as demonstrated by L101V and V188I changes in the L3 protein amino acid sequences. The high frequency in RTE food of MDR-CoNS including methicillin-resistant (MR-CoNS) strains constitutes a direct risk to public health as they increase the gene pool from which pathogenic bacteria can pick up resistance traits.

Single nucleotide polymorphisms of intervertebral disc collagens and prospects for their correction

The main functions of the intervertebral disc (IVD) are ensured by the reliable integration of three structures of the IVD: the annulus fibrosus (AF), the hydrated nucleus pulposus (NP) and the two cartilaginous end plates (CEP). All molecular components are involved in the integration of the three anatomical structures of the IVD, however, the most important biomechanical properties - resistance to rupture / stretching / shift, resistance to static axial loads are mostly determined by collagens.The unique properties of collagens depend on the amino acid sequence of the three alpha (α) chains, which, after spiralization and condensation, form the collagen molecule - tropocollagen. The amino acid sequence contains all the necessary information for spiralization, modification, secretion of tropocollagen, its processing, condensation into fibrils and fibers according to the self-assembly principle. Changes in the primary amino acid sequence, depending on the substitution itself and its localization, lead to disruption of the stages of tropocollagen formation, its extracellular processing, and condensation.Currently, most of the research is devoted to the study of polymorphisms in the genes of IVD collagen types I, II, IX and XI. Algorithms for using information about genetic polymorphisms of collagen genes are only being formed. Data on genetic variation are often conflicting. An important aspect is the homogeneity of the study group by age, ethnicity, gender, as well as by the type of degenerative changes. There is also insufficient data on the effect of polymorphism on the properties of the collagen molecule, which greatly complicates the creation of standards for therapeutic correction.This literature review is devoted to the consideration of new data on collagen genes polymorphisms, the impact of these polymorphisms on integrative relationships in IVD structures, as well as the prospects for the correction of genetic abnormalities.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition

Uridine diphosphate-dependent glycosyltransferases (UGTs) mediate the glycosylation of plant metabolites, thereby altering their physicochemical properties and bioactivities. Plants possess numerous UGT genes, with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics, but the biological function and molecular basis of these phenomena are not fully understood. The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition (Ki) at 4 μM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 μM. We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs. A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reduced scopoletin substrate inhibition, whereas its monolignol glycosylation activity was almost unaffected. Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation, leading to enhanced promiscuity, which was accompanied by substrate inhibition. Studies of 3D structures identified open and closed UGT conformers, allowing visualization of the dynamics of conformational changes that occur during catalysis. Previously postulated substrate access tunnels likely serve as drainage channels. The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release. Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions. The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

Competitive evolution of H1N1 and H3N2 influenza viruses in the United States: A mathematical modeling study

Seasonal influenza causes vast public health and economic impact globally. The prevention and control of the annual epidemics remain a challenge due to the antigenic evolution of the viruses. Here, we presented a novel modeling framework based on changes in amino acid sequences and relevant epidemiological data to retrospectively investigate the competitive evolution and transmission of H1N1 and H3N2 influenza viruses in the United States during October 2002 and April 2019. To do so, we estimated the time-varying disease transmission rate from the reported influenza cases and the time-varying antigenic change rate of the viruses from the changes in amino acid sequences. By incorporating the time-varying antigenic change rate into the transmission models, we found that the models could capture the evolutionary transmission dynamics of influenza viruses in the United States. Our modeling results also showed that the antigenic change of the virus plays an essential role in seasonal influenza dynamics.

What a Difference an Amino Acid Makes: An All-Atom Simulation Study of Nonameric Peptides in Inhibitory HLA-E/NKG2A/CD94 Immune Complexes.

MHC class I antigen E (HLA-E), a ligand for the inhibitory NKG2A/CD94 receptor of the immune system, is responsible for evading the immune surveillance in several settings, including senescent cell accumulation and tumor persistence. The formation of this ligand-receptor interaction promotes the inhibition of the cytolytic action of immune system natural killer (NK) cells and CD8+ T-cells expressing this receptor. The final outcome of the HLA-E/NKG2A/CD94 interaction on target cells is also highly dependent on the identity of the nonameric peptide incorporated into the HLA-E ligand. To better understand the role played by a nonameric peptide in these immune complexes, we performed a series of multi-microsecond all-atom molecular dynamics simulations. We generated natural and alternative variants of the nonameric peptide bound to the HLA-E ligand alone or in the HLA-E/NKG2A/CD94 complexes. A systematic study of molecular recognition between HLA-E and peptides led to the development of new variants that differ at the strategic 6th position (P6) of the peptide and have favorable in silico properties comparable to those of natural binding peptides. Further examination of a selected subset of peptides in full complexes revealed a new variant that, according to our previously derived atomistic model, can interfere with the signal transduction via HLA-E/NKG2A/CD94 and thus prevent the target cell from evading immune clearance by NK and CD8+ T-cells. These simulations provide an atomistic picture of how a small change in amino acid sequence can lead to a profound effect on binding and molecular recognition. Furthermore, our study also provides new data on the peptide interaction motifs as well as the energetic and conformational properties of the binding interface, laying the structure-based foundation for future development of potential therapeutic peptides, peptidomimetics, or even small molecules that would bind to the HLA-E ligand and abrogate NKG2A/CD94 recognition. Such external intervention would be useful in the emerging field of targeting senescent cells in a variety of age-related diseases, as well as in novel cancer immunotherapies.

Molecular characterization of α-lactalbumin (LALBA) protein in Indian buffalo(Bubalus bubalis)

The full open reading frame (ORF) of the buffalo beta-lactoglobulin (Bu_LALBA) gene was characterized. Results showed that Bu_LALBA ORF consisted of 429 bp long (142 aa residues) with three nucleotide variations at 111bp, 147 bp, and 291 bp but no change in amino acid sequence. The MSA showed that the Bu_LALBA was more different from the pig and human LALBA sequences. The phylogenetic tree showed that the cow, yak, and buffalo formed one cluster, while the buffalo was more closer (94%) to domestic yak (Bos grunniens). Results of ExPASy analysis showed that the Bu_LALBA protein was acidic (pI, 4.81), thermo-tolerant, and hydrophilic. However, the presence of random coil (33.80%) and α-helix (41.5%) in Bu_LALBA protein suggest that the protein was flexible and thermostable. Thirty liner motifs were identified, indicating that the Bu_LALBA act as regulatory protein. The tertiary structure of Bu_LALBA predicted by I-TASSER showed a more stabilized nature of LALBA protein. Further, the Ramachandran plot validated the 3-D structure of Bu_LALBA, which was of decent quality. The presence of four ligand-binding sites in Bu_LALBA (calcium ion, glycine, N-acetyl-L-glutamate, and N-acetylglucosamine) proposed that the LALBA binds to several fatty acids and ions. The presence of four serine, four threonine, two tyrosine residues, and six methylated lysine and five acetyl-lysine sites in Bu_LALBA indicated that the protein was involved in post-translational modification processes. IEDB analysis showed the presence of five and one epitope sites in Bu_LALBA protein for B-cell and T-cell, respectively, which suggest that this protein has certain immunological roles.

Identification of SSTR5 Gene Polymorphisms and Their Association With Growth Traits in Hulun Buir Sheep.

The aim of this study was to locate SSTR5 polymorphisms and evaluate their association with growth traits in Hulun Buir sheep. The study followed up 884 Hulun Buir sheep from birth to 16 months of age, which were born in the same pasture and the same year, and a consistent grazing management strategy was maintained. The birth weight (BRW) was recorded at birth, and body weight (BW), body height (BH), body length (BL), chest circumference (ChC), chest depth (ChD), chest width (ChW), hip width (HW), and cannon circumference (CaC) were measured at 4 and 9 months of age. BW, BH, BL, ChD, HW, and CaC were also recorded at 16 months of age. Based on the growth traits, 233 sheep were selected as experimental animals. Sanger sequencing was performed, and seven single-nucleotide polymorphisms (SNPs) were identified. Association analyses of the SNPs and the growth traits were then conducted. Seven SNPs of the SSTR5 exhibited moderate polymorphism (0.25<PIC<0.5) and were consistent with the Hardy–Weinberg equilibrium. SNP7 (T989C, rs601836309) caused a change in amino acid sequences, while others did not cause any change. The genotypes of SNP1 (C186T, s400914340) were significantly associated with BW, ChW, and ChC at 4 months of age and with HW at 9 months of age (p<0.05). These genotypes also showed extremely significant association with CaC at 4 months of age (p<0.01). The genotypes of SNP7 exhibited a significant association with ChW and CaC at 4 and 9 months of age, respectively. Moreover, the genotypes of SNP3 (T384C, rs413380618)) and SNP4 (T537C, rs605867745) were significantly associated with CaC at 9 months of age (p<0.05). Linkage disequilibrium was observed among the seven SNPs with five haplotypes. However, these haplotypes were not associated with growth traits at different ages. In conclusion, SNP1, SNP3, SNP4, and SNP7 may serve as molecular markers for the growth traits of Hulun Buir sheep.

Note on DNA Analysis and Redesigning Using Markov Chain.

The paper contains a discussion on mathematical modifying and redesigning DNA with the use of Markov chains. We give a simple mathematical technique for overwriting missing parts of DNA. With a certain probability (without even knowing the function of the missing codon) we can find a synonymous codon, so that there is no frequency change in amino acid sequences of proteins. We use Markov Chain to analyze the dependencies in DNA sequence of the human gene Alpha 1,3-Galactosyltransfe rase 2. We include a theoretical introduction which facilitates the understanding of the paper for non-mathematicians, especially for biologists not familiar with the theory of Markov chains.

Pepducin-mediated G Protein-Coupled Receptor Signaling in the Cardiovascular System.

Pepducins are small-lipidated peptides designed from the intracellular loops of G protein-coupled receptors (GPCRs) that act in an allosteric manner to modulate the activity of GPCRs. Over the past 2 decades, pepducins have progressed initially from pharmacologic tools used to manipulate GPCR activity in an orthosteric site-independent manner to compounds with therapeutic potential that have even been used safely in phase 1 and 2 clinical trials in human subjects. The effect of pepducins at their cognate receptors has been shown to vary between antagonist, partial agonist, and biased agonist outcomes in various primary and clonal cell systems, with even small changes in amino acid sequence altering these properties and their receptor selectivity. To date, pepducins designed from numerous GPCRs have been studied for their impact on pathologic conditions, including cardiovascular diseases such as thrombosis, myocardial infarction, and atherosclerosis. This review will focus in particular on pepducins designed from protease-activated receptors, C-X-C motif chemokine receptors, formyl peptide receptors, and the β2-adrenergic receptor. We will discuss the historic context of pepducin development for each receptor, as well as the structural, signaling, pathophysiologic consequences, and therapeutic potential for each pepducin class.

Characterization of alternatively spliced transcript variants of glycophorin A and glycophorin B genes in Chinese blood donors.

The molecular basis of MNS blood group variants is not fully clear yet. In this study, we have characterized mRNA variants of GYPA and GYPB genes to reveal whether alternative RNA splicing may cause antigenic diversity of the MNS system. Total RNA was extracted from peripheral blood of Chinese blood donors and full-length cDNA products were generated. A nested polymerase chain reaction (PCR)-based method was established for fragment amplification and Sanger sequencing. Resulted full-length mRNA sequences were aligned with GYPA or GYPB genomic sequences respectively for exon identification. Amino acid (AA) sequences of GPA and GPB proteins were extrapolated and GYPA-EGFP, GYPB-EGFP fusion genes were generated to monitor subcellular distribution of the encoded glycophorin (GP) proteins. Totally 10 blood samples were analysed. GYPB mRNAs of all the subjects demonstrated frequent exon insertion or deletion whereas this kind of variation was only observed in 3 of 10 GYPA mRNA samples. None of the reported Miltenberger hybrids was detected in any of the mRNA samples. The alternative splicing resulted in changes of AA sequences in N-terminal domains where the MNS antigenic motifs resided; however, subcellular localizations of GP-EGFP fusion proteins showed that the above-mentioned AA changes did not affect cell surface distribution of the encoded GP proteins. Alternative RNA splicing may influence the antigenic features of GP proteins but not their cell surface distribution. Therefore, GYPA and GYPB mRNA characterization might be an invaluable supplement to serological phenotyping and DNA-based genotyping in MNS blood grouping.

A commentary of “The brain evolutionary mechanism of feeding preference”: 10 remarkable discoveries from 2020 in Nature

The fly Drosophila sechellia feeds exclusively on the toxic noni fruit (Morinda citrifolia). What makes this species such a picky eater compared with its generalist relatives? Auer et al. cracked the case using the genome-editing tool CRISPR/Cas9 [1]. One sensory neuron class expressing the odorant receptor 22a (Or22a) protein is more abundant in D. sechellia than in other fly species. The group established that small changes in Or22a's amino-acid sequence have contributed to D. sechellia’s preference for noni. They also identified several other evolutionary changes that might contribute to this apparently simple behavioral shift. Even tiny flies that love stinky fruit can provide powerful insight into how brains evolve to shape complex behaviors.①①Original source in Chinese: Yufeng Pan, The brain evolutionary mechanism of feeding preference, Bulletin of National Natural Science Foundation of China. 35 (2) (2021) 242–243.

Computational study of the impact of nucleotide variations on highly conserved proteins: In the case of actin.

Sequencing of individual human genomes enables studying relationship among nucleotide variations, amino acid substitutions, effect on protein structures and diseases. Many studies have found general tendencies, for instance, that pathogenic variations tend to be found in the buried regions of the protein structures, that benign variations tend to be found on the surface of the proteins, and that variations on evolutionary conserved residues tend to be pathogenic. These tendencies were deduced from globular proteins with standard evolutionary changes in amino acid sequences. In this study, we investigated the variation distribution on actin, one of the highly conserved proteins. Many nucleotide variations and three-dimensional structures of actin have been registered in databases. By combining those data, we found that variations buried inside the protein were rather benign and variations on the surface of the protein were pathogenic. This idiosyncratic distribution of the variation impact is likely ascribed to the extensive use of the surface of the protein for protein-protein interactions in actin.