Technical considerations in Hi-C scaffolding and evaluation of chromosome-scale genome assemblies.

The recent development of ecological studies has been fueled by the introduction of massive information based on chromosome-scale genome sequences, even for species for which genetic linkage is not accessible. This was enabled mainly by the application of Hi-C, a method for genome-wide chromosome conformation capture that was originally developed for investigating the long-range interaction of chromatins. Performing genomic scaffolding using Hi-C data is highly resource-demanding and employs elaborate laboratory steps for sample preparation. It starts with building a primary genome sequence assembly as an input, which is followed by computation for genome scaffolding using Hi-C data, requiring careful validation. This article presents technical considerations for obtaining optimal Hi-C scaffolding results and provides a test case of its application to a reptile species, the Madagascar ground gecko (Paroedura picta). Among the metrics that are frequently used for evaluating scaffolding results, we investigate the validity of the completeness assessment of chromosome-scale genome assemblies using single-copy reference orthologues.

The BLOC-3 subunit HPS4 is required for activation of Rab32/38 GTPases in melanogenesis, but its Rab9 activity is dispensable for melanogenesis.

HPS4 biogenesis of lysosome-related organelles complex 3 subunit 2 (HPS4) is one of the genes whose mutations have been associated with Hermansky-Pudlak syndrome (HPS), characterized by ocular albinism and susceptibility to bleeding because of defects in the biogenesis of lysosome-related organelles such as melanosomes. HPS4 protein forms a BLOC-3 complex with HPS1, another HPS gene product, and the complex has been proposed to function as a guanine nucleotide exchange factor (GEF) for RAB32, a member of the Rab small GTPase family (Rab32), and Rab38 (Rab32/38-GEF) and also as a Rab9 effector. Although both Rab32/38 and Rab9 have been shown previously to be involved in melanogenesis in mammalian epidermal melanocytes, the functional relationships of these small GTPases with BLOC-3 remain unknown. In this study, we used site-directed mutagenesis to generate HPS4 mutants that specifically lack either Rab32/38-GEF activity or Rab9-binding activity and investigated their involvement in melanogenesis of melan-le cells (an HPS4-deficient melanocyte cell line derived from light ear mice). Melan-le cells exhibit a clear hypopigmentation phenotype, i.e. reduced expression and abnormal distribution of tyrosinase and reduced melanin content. Although re-expression of WT HPS4 completely rescued this phenotype, the Rab32/38-GEF activity-deficient HPS4 mutant failed to restore melanin content and tyrosinase trafficking in these cells. Unexpectedly, as WT HPS4, the Rab9 binding-deficient HPS4 mutant completely rescued the phenotype. These results indicate that activation of Rab32/38 by HPS4 (or BLOC-3) is essential for melanogenesis of cultured melanocytes and that Rab9 likely regulates melanogenesis independently of HPS4.

Different effects of crizotinib treatment in two non-small cell lung cancer patients with SDC4::ROS1 fusion variants.

The possibility of stratifying patients according to differences in ROS proto-oncogene 1 (ROS1) fusion partners has been discussed. This study aimed to clarify the clinicopathological differences between two SDC4::ROS1 positive NSCLC cases who had different responses to crizotinib. Cytology and pathology samples from two NSCLC cases with SDC4::ROS1 who were diagnosed and treated with crizotinib at Nihon University Itabashi Hospital were obtained. Case 1 has been well-controlled with crizotinib for over 5 years, but case 2 was worse and overall survival was 19 months. Sequencing analysis of ROS1 fusion genes was performed by reverse-transcription-PCR and Sanger's sequencing methods. In addition, thyroid transcription factor (TTF)-1, ROS-1, Ki67, and phosphorylated extracellular signal-regulated kinase (pERK)1/2 expression were investigated using immunohistochemistry. Sequencing analysis showed SDC4 exon2::ROS1 exon 32 (exon33 deleted) in case 1, and coexistence of SDC4 exon2::ROS1 exon 34 and SDC4 exon2::ROS1 exon35 in case 2. The Ki67 index was not different, but ROS1 and pERK1/2 expression levels tended to be higher in the tumor cells of case 2 than in case 1. Therapeutic response to crizotinib and patients' prognosis in ROS1 rearranged NSCLC may be related to the activation of ROS1 signaling, depending on ROS1 and pERK1/2 overexpression status, even if the ROS1 fusion partner is the same.

Egg Yolk Protein Homologs Identified in Live-Bearing Sharks: Co-Opted in the Lecithotrophy-to-Matrotrophy Shift?

Reproductive modes of vertebrates are classified into two major embryonic nutritional types: yolk deposits (i.e., lecithotrophy) and maternal investment (i.e., matrotrophy). Vitellogenin (VTG), a major egg yolk protein synthesized in the female liver, is one of the molecules relevant to the lecithotrophy-to-matrotrophy shift in bony vertebrates. In mammals, all VTG genes are lost following the lecithotrophy-to-matrotrophy shift, and it remains to be elucidated whether the lecithotrophy-to-matrotrophy shift in nonmammalians is also associated with VTG repertoire modification. In this study, we focused on chondrichthyans (cartilaginous fishes)-a vertebrate clade that underwent multiple lecithotrophy-to-matrotrophy shifts. For an exhaustive search of homologs, we performed tissue-by-tissue transcriptome sequencing for two viviparous chondrichthyans, the frilled shark Chlamydoselachus anguineus and the spotless smooth-hound Mustelus griseus, and inferred the molecular phylogeny of VTG and its receptor very low-density lipoprotein receptor (VLDLR), across diverse vertebrates. As a result, we identified either three or four VTG orthologs in chondrichthyans including viviparous species. We also showed that chondrichthyans had two additional VLDLR orthologs previously unrecognized in their unique lineage (designated as VLDLRc2 and VLDLRc3). Notably, VTG gene expression patterns differed in the species studied depending on their reproductive mode; VTGs are broadly expressed in multiple tissues, including the uterus, in the two viviparous sharks, and in addition to the liver. This finding suggests that the chondrichthyans VTGs do not only function as the yolk nutrient but also as the matrotrophic factor. Altogether, our study indicates that the lecithotrophy-to-matrotrophy shift in chondrichthyans was achieved through a distinct evolutionary process from mammals.

Squalomix: shark and ray genome analysis consortium and its data sharing platform.

The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info.

Griscelli Syndrome Type 2 Sine Albinism: Unraveling Differential RAB27A Effector Engagement.

Griscelli syndrome type 2 (GS-2) is an inborn error of immunity characterized by partial albinism and episodes of hemophagocytic lymphohistiocytosis (HLH). It is caused by RAB27A mutations that encode RAB27A, a member of the Rab GTPase family. RAB27A is expressed in many tissues and regulates vesicular transport and organelle dynamics. Occasionally, GS-2 patients with RAB27A mutation display normal pigmentation. The study of such variants provides the opportunity to map distinct binding sites for tissue-specific effectors on RAB27A. Here we present a new case of GS-2 without albinism (GS-2 sine albinism) caused by a novel missense mutation (Val143Ala) in the RAB27A and characterize its functional cellular consequences. Using pertinent animal cell lines, the Val143Ala mutation impairs both the RAB27A-SLP2-A interaction and RAB27A-MUNC13-4 interaction, but it does not affect the RAB27A-melanophilin (MLPH)/SLAC2-A interaction that is crucial for skin and hair pigmentation. We conclude that disruption of the RAB27A-MUNC13-4 interaction in cytotoxic lymphocytes leads to the HLH predisposition of the GS-2 patient with the Val143Ala mutation. Finally, we include a review of GS-2 sine albinism cases reported in the literature, summarizing their genetic and clinical characteristics.