-
Biomolecules May 2024Through machine learning, identifying correlations between amino acid sequences of antibodies and their observed characteristics, we developed an internal viscosity...
Through machine learning, identifying correlations between amino acid sequences of antibodies and their observed characteristics, we developed an internal viscosity prediction model to empower the rapid engineering of therapeutic antibody candidates. For a highly viscous anti-IL-13 monoclonal antibody, we used a structure-based rational design strategy to generate a list of variants that were hypothesized to mitigate viscosity. Our viscosity prediction tool was then used as a screen to cull virtually engineered variants with a probability of high viscosity while advancing those with a probability of low viscosity to production and testing. By combining the rational design engineering strategy with the in silico viscosity prediction screening step, we were able to efficiently improve the highly viscous anti-IL-13 candidate, successfully decreasing the viscosity at 150 mg/mL from 34 cP to 13 cP in a panel of 16 variants.
Topics: Viscosity; Protein Engineering; Antibodies, Monoclonal; Machine Learning; Amino Acid Sequence; Humans
PubMed: 38927021
DOI: 10.3390/biom14060617 -
Genome Biology Jun 2024Variable number tandem repeats (VNTRs) are highly polymorphic DNA regions harboring many potentially disease-causing variants. However, VNTRs often appear unresolved...
BACKGROUND
Variable number tandem repeats (VNTRs) are highly polymorphic DNA regions harboring many potentially disease-causing variants. However, VNTRs often appear unresolved ("dark") in variation databases due to their repetitive nature. One particularly complex and medically relevant VNTR is the KIV-2 VNTR located in the cardiovascular disease gene LPA which encompasses up to 70% of the coding sequence.
RESULTS
Using the highly complex LPA gene as a model, we develop a computational approach to resolve intra-repeat variation in VNTRs from largely available short-read sequencing data. We apply the approach to six protein-coding VNTRs in 2504 samples from the 1000 Genomes Project and developed an optimized method for the LPA KIV-2 VNTR that discriminates the confounding KIV-2 subtypes upfront. This results in an F1-score improvement of up to 2.1-fold compared to previously published strategies. Finally, we analyze the LPA VNTR in > 199,000 UK Biobank samples, detecting > 700 KIV-2 mutations. This approach successfully reveals new strong Lp(a)-lowering effects for KIV-2 variants, with protective effect against coronary artery disease, and also validated previous findings based on tagging SNPs.
CONCLUSIONS
Our approach paves the way for reliable variant detection in VNTRs at scale and we show that it is transferable to other dark regions, which will help unlock medical information hidden in VNTRs.
Topics: Minisatellite Repeats; Humans; Cardiovascular Diseases; Genetic Variation; Sequence Analysis, DNA; Lipoprotein(a); Genetic Predisposition to Disease
PubMed: 38926899
DOI: 10.1186/s13059-024-03316-5 -
BMC Biotechnology Jun 2024Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with...
BACKGROUND
Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS
In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS
The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS
This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Topics: Animals; CHO Cells; Integrases; Cricetulus; Nuclear Localization Signals; Cell Nucleus; Serine; Green Fluorescent Proteins; Cricetinae; Xenopus laevis
PubMed: 38926833
DOI: 10.1186/s12896-024-00871-4 -
BMC Plant Biology Jun 2024Delphinium L. represents a taxonomically intricate genus of significant phylogenetic and economic importance in Ranunculaceae. Despite the existence of few chloroplast... (Comparative Study)
Comparative Study
Complete chloroplast genomes of eight Delphinium taxa (Ranunculaceae) endemic to Xinjiang, China: insights into genome structure, comparative analysis, and phylogenetic relationships.
BACKGROUND
Delphinium L. represents a taxonomically intricate genus of significant phylogenetic and economic importance in Ranunculaceae. Despite the existence of few chloroplast genome datasets, a comprehensive understanding of genome structures and selective pressures within the genus remains unknown. Furthermore, several taxa in this genus are exclusively found in Xinjiang, China, a region renowned for its distribution and diversity of Chinese and Central Asian Delphinium species. Therefore, investigating the features of chloroplast genomes in this area will provide valuable insights into the evolutionary processes and phylogenetic relationships of the genus.
RESULTS
In this study, the eight newly completed chloroplast genomes are examined, ranging in length from 153,979 bp to 154,284 bp. Alongside these, analysing six previously reported taxa re-annotated in Delphinium, 111 unique genes are identified across all samples. Genome structure, distributions of simple sequence repeats and short dispersed repeats, as well as gene content are similar among these Delphinium taxa. Nine hypervariable intergenic spacers and protein coding regions, including ndhF-trnL, rpl16-intron, rpl33, rps15, rps18, trnK-trnQ, trnP-psaJ, trnT-psbD and ycf1, are identified among 13 perennial Delphinium. Selective pressure and codon usage bias of all the plastid genes are performed within 14 Delphinium taxa. Phylogenetic analysis based on 14 Delphinium plastomes, alongside two Aconitum (Ranunculaceae) species serving as outgroup taxa, reveals the monophyletic nature of Delphinium. Our findings further discern Delphinium into two distinct clades: perennial species (clade I) and annual species (clade II). In addition, compared with the nrDNA ITS topology, cytological data and morphological characters, D. mollifolium and D. maackianum showed potential involvement in hybridization or polyploidization processes. Excluding these two species, the perennial Delphinium (clade I) exhibits a stronger consistency with the morphology-based system that utilized seed morphology.
CONCLUSION
This study represents the first comprehensive analysis of plastomic variations among Delphinium taxa, based on the examination of 14 complete plastomes. The chloroplast genome structure of Delphinium is similar to other angiosperms and possesses the typical quadripartite structure with the conserved genome arrangement and gene features. In addition, the variation of non-coding regions is larger than coding regions of the chloroplast genome. Through DNA sequence divergence across Delphinium plastomes and subsequent phylogenomic analyses ndhF-trnL and ycf1 are identified as promising molecular markers. These highly variable loci held significant potential for future phylogenetic and phylogeographic studies on Delphinium. Our phylogenomic analyses based on the whole plastomes, concatenation of 132 unique intergenic spacer regions, concatenation of 77 unique protein-coding genes and nrDNA ITS, all support the monophyly of Delphinium and perennial taxa clusters together into one clade within this genus. These findings provide crucial data for systematic, phylogenomic and evolutionary research in the genus for future studies.
Topics: Genome, Chloroplast; Phylogeny; Delphinium; China; Ranunculaceae
PubMed: 38926811
DOI: 10.1186/s12870-024-05279-y -
Journal of Translational Medicine Jun 2024Existing studies have found that circular RNAs (circRNAs) act as sponges for micro RNAs (miRNAs) to control downstream genes. However, the specific functionalities and...
BACKGROUND
Existing studies have found that circular RNAs (circRNAs) act as sponges for micro RNAs (miRNAs) to control downstream genes. However, the specific functionalities and mechanisms of circRNAs in human clear cell renal cell carcinoma (ccRCC) have yet to be thoroughly investigated.
METHODS
Patient cohorts from online databases were used to screen candidate circRNAs, while another cohort from our hospital was obtained for validation. CircSOD2 was identified as a potential oncogenic target, and its relevant characteristics were investigated during ccRCC progression through various assays. A positive feedback loop containing downstream miRNA and its target gene were identified using bioinformatics and validated by luciferase reporter assays, RNA pull-down, and high-throughput sequencing.
RESULTS
CircSOD2 expression was elevated in tumor samples and significantly correlated with overall survival (OS) and the tumor stage of ccRCC patients, which appeared in the enhanced proliferation, invasion, and migration of tumor cells. Through competitive binding to circSOD2, miR-532-3p can promote the expression of PAX5 and the progression of ccRCC, and such regulation can be salvaged by miR-532-3p inhibitor.
CONCLUSION
A novel positive feedback loop, PAX5/circSOD2/miR-532-3p/PAX5 was identified in the study, indicating that the loop may play an important role in the diagnosis and prognostic prediction in ccRCC patients.
Topics: Humans; Carcinoma, Renal Cell; RNA, Circular; Kidney Neoplasms; MicroRNAs; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Cell Line, Tumor; Cell Proliferation; Female; Middle Aged; Male; Carcinogenesis; Cell Movement; PAX5 Transcription Factor; Oncogenes; Base Sequence; Disease Progression; Neoplasm Invasiveness; Reproducibility of Results
PubMed: 38926764
DOI: 10.1186/s12967-024-05290-9 -
Scientific Reports Jun 2024To analyse the genetic aetiology of a child with oculocutaneous albinism and to explore the effects of two mutation sites on the function of the OCA2 protein at the mRNA...
To analyse the genetic aetiology of a child with oculocutaneous albinism and to explore the effects of two mutation sites on the function of the OCA2 protein at the mRNA and protein levels via the use of recombinant carriers in vitro. Whole-exome sequencing (WES) and Sanger sequencing were used to analyse the pathogenic genes of the child and validate the mutations in the parents. pEGFP and phage vectors carrying wild-type and mutant OCA2 were constructed using the coding DNA sequence (CDS) of the whole gene-synthesized OCA2 as a template and transfected into HEK293T cells, after which expression analysis was performed. The child in this study was born with white skin, hair, eyelashes, and eyebrows and exhibited nystagmus. Genetic analysis indicated that the child carried two heterozygous mutations: c.1079C > T (p.Ser360Phe) of maternal origin and c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) of paternal origin, conforming to an autosomal recessive inheritance pattern. In vitro analysis showed that the expression of the c.1079C > T (p.Ser360Phe) mutant did not significantly change at the mRNA level but did increase at the protein level, suggesting that the mutation may lead to enhanced protein stability, and the c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) mutation resulted in the loss of three amino acids in exon 10, producing a truncated protein. In vitro expression analysis also revealed that the expression of the mutant gene was significantly downregulated at both the mRNA and protein levels, suggesting that the mutation can simultaneously produce truncated proteins and lead to protein degradation. This case study enriches the phenotypic spectrum of OCA2 gene disease. In vitro expression analysis confirmed that both mutations affect protein expression, providing a theoretical basis for analysing the pathogenicity of these two mutations.
Topics: Humans; HEK293 Cells; Mutation; Albinism, Oculocutaneous; Membrane Transport Proteins; Exome Sequencing; Female; Male; Pedigree; RNA, Messenger
PubMed: 38926510
DOI: 10.1038/s41598-024-64782-2 -
Life Science Alliance Sep 2024In , inter-cellular transport of the small non-coding RNA causing systemic RNAi is mediated by the transmembrane protein SID1, encoded by the gene in the systemic RNAi...
In , inter-cellular transport of the small non-coding RNA causing systemic RNAi is mediated by the transmembrane protein SID1, encoded by the gene in the systemic RNAi defective () loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the ChUP family. Although systemic RNAi is not an evolutionarily conserved process, the gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of gene products-hSIDT1 and hSIDT2-mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra-cytosolic domain of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain exists as two modules-a flexible lipid binding domain and a rigid transmembrane domain core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in ChUP family members.
Topics: Humans; Membrane Proteins; Protein Binding; Protein Domains; Models, Molecular; Protein Conformation; Caenorhabditis elegans; Animals; Amino Acid Sequence; Binding Sites; Lipids; Caenorhabditis elegans Proteins; RNA Interference
PubMed: 38925866
DOI: 10.26508/lsa.202402624 -
Science Advances Jun 2024Mutations in cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is...
Mutations in cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, . We show that >50% of short RNA-sequencing reads mapping to also map to . Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both and . We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both and . These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function-yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for and with implications for our understanding of the role of in health and disease.
Topics: Humans; Glucosylceramidase; Pseudogenes; Brain; Molecular Sequence Annotation; Parkinson Disease; Gaucher Disease; Sequence Analysis, RNA
PubMed: 38924406
DOI: 10.1126/sciadv.adk1296 -
Plant Biotechnology Journal Jun 2024Developing early maturing lentil has the potential to minimize yield losses, mainly during terminal drought. Whole-genome resequencing (WGRS) based QTL-seq identified...
Developing early maturing lentil has the potential to minimize yield losses, mainly during terminal drought. Whole-genome resequencing (WGRS) based QTL-seq identified the loci governing earliness in lentil. The genetic analysis for maturity duration provided a good fit to 3:1 segregation (F), indicating earliness as a recessive trait. WGRS of Globe Mutant (late parent), late-flowering, and early-flowering bulks (from RILs) has generated 1124.57, 1052.24 million raw and clean reads, respectively. The QTL-Seq identified three QTLs (LcqDTF3.1, LcqDTF3.2, and LcqDTF3.3) on chromosome 3 having 246244 SNPs and 15577 insertions/deletions (InDels) and 13 flowering pathway genes. Of these, 11 exhibited sequence variations between bulks and validation (qPCR) revealed a significant difference in the expression of nine candidate genes (LcGA20oxG, LcFRI, LcLFY, LcSPL13a, Lcu.2RBY.3g060720, Lcu.2RBY.3g062540, Lcu.2RBY.3g062760, LcELF3a, and LcEMF1). Interestingly, the LcELF3a gene showed significantly higher expression in late-flowering genotype and exhibited substantial involvement in promoting lateness. Subsequently, an InDel marker (I-SP-383.9; LcELF3a gene) developed from LcqDTF3.2 QTL region showed 82.35% PVE (phenotypic variation explained) for earliness. The cloning, sequencing, and comparative analysis of the LcELF3a gene from both parents revealed 23 SNPs and InDels. Interestingly, a 52 bp deletion was recorded in the LcELF3a gene of L4775, predicted to cause premature termination of protein synthesis after 4 missense amino acids beyond the 351st amino acid due to the frameshift during translation. The identified InDel marker holds significant potential for breeding early maturing lentil varieties.
PubMed: 38923713
DOI: 10.1111/pbi.14415 -
Cell Reports Jun 2024
PubMed: 38923456
DOI: 10.1016/j.celrep.2024.114418