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The human major histocompatibility complex (MHC) is a ∼4 Mb genomic segment on Chromosome 6 that plays a pivotal role in the immune response. Despite its importance in various traits and diseases, its complex nature makes it challenging to accurately characterize on a routine basis. We present a novel approach allowing targeted sequencing and de novo haplotypic assembly of the MHC region in heterozygous samples, using long-read sequencing technologies. Our approach is validated using two reference samples, two family trios, and an African-American sample. We achieved excellent coverage (96.6%–99.9% with at least 30× depth) and high accuracy (99.89%–99.99%) for the different haplotypes. This methodology offers a reliable and cost-effective method for sequencing and fully characterizing the MHC without the need for whole-genome sequencing, facilitating broader studies on this important genomic segment and having significant implications in immunology, genetics, and medicine.

The technical limitations of current next-generation sequencing technologies, combined with an ever-increasing number of human leukocyte antigen (HLA) alleles, form the basis for the additional ambiguities encountered at an increasing rate in clinical practice. HLA-DPB1characterization, particularly, generates a significant percentage of ambiguities (25.5%), posing a challenge for accurate and unambiguous HLA-DPB1genotyping. Phasing of exonic heterozygous positions between exon 2 and all other downstream exons has been the major cause of ambiguities. In this study, the Oxford Nanopore MinION, a third-generation sequencing technology, was used to resolve the phasing. The accurate MiSeq sequencing data, combined with the long reads obtained from the MinION platform, allow for the resolution of the tested ambiguities.

Sclerotinia sclerotiorum differentiates by forming terminal-type sclerotia. We found that the fungus produces β-carotene in low and high levels during its undifferentiated and differentiated stages, respectively, and at low (in dark) and high (in light) oxidative growth conditions, respectively. In contrast, a sclerotium non-producing strain of S. sclerotiorum produces very low amounts of β-carotene in constant levels throughout its entire growth period, irrespective of oxidative stress degree of growth conditions. When β-carotene was added to the growth medium of the sclerotium producing strain, it caused a concentration-dependent inhibition of sclerotia formation. We propose that β-carotene inhibits sclerotial differentiation in S. sclerotiorum by lowering oxidative stress via its antioxidant scavenging properties.

Retinoic acid receptor-β (RAR-β) specifically binds retinoic acid (RA) and functions as a RA-inducible transcriptional regulatory factor. Simultaneous mutation of Arg269and Lys220of RAR-β to Ala results in a dramatic reduction in both transactivation and affinity for RA along with creating a RA concentration-dependent dominant negative mutant. In this report, we found that mutation of these two amino acid residues singly and simultaneously to Gln results in mutant RAR-βs, each displaying a more dramatic reduction in transactivation and affinity for RA than their corresponding Ala mutant, with the R269Q more profoundly affected than K220Q. Furthermore, we examined both the Ala and Gln mutants for their ability to transactivate and bind two other retinoids with different functional end groups (all-trans-retinol and all-trans-retinal). Mutation of Lys220to either an Ala or a Gln favors transactivation and binding of retinal, while mutation of either Lys220or Arg269to Gln favors retinol transactivation and binding. Taken together, these results suggest that Arg269and Lys220lie within the ligand binding pocket of RAR-β and that these two amino acid residues play an important role in determining retinoid specificity most likely by directly interacting with the carboxylate group of RA.