Nuclear Gene Research Articles

Identification of orange color-related gene, PhcpcC, in Pyropia haitanensis

Pigmentation-related mutations can be utilized to distinguish between differentially colored sectors of chimeric thalli, thereby facilitating the efficient breeding of economically valuable Pyropia/Porphyra seaweed species. However, the specific trait loci and alleles responsible for Pyropia/Porphyra coloration have yet to be identified, which limits the applicability of coloration mutants for breeding and genetic analyses. In this study, to preserve the genetic integrity of the population, only four-colored thalli were considered when constructing the doubled haploid (DH) Pyropia haitanensis population, which consisted of 480 homozygous offspring lines (representing the largest DH Pyropia/Porphyra population). The offspring lines in the DH population exhibited both wild-type colored and orange sectors, with a segregation ratio of approximately 1:1, indicating that the orange coloration was controlled by a single nuclear gene. Through BSA-seq analysis (99% confidence interval), a candidate region of 0.5 Mb was identified in the P. haitanensis genome. Additionally, a non-synonymous SNP [A/G] was detected at base-pair position 481 in the coding region of PhcpcC, which encodes a phycocyanin-associated rod linker protein. This SNP locus was verified in both DH and natural populations, with the wild-type colored lines having an A base and the orange lines having a G base at this locus. Therefore, PhcpcC may be the gene associated with the orange coloration of P. haitanensis. The molecular marker developed in this study can be employed to exploit pigmentation mutants for breeding and genetic analyses of Pyropia/Porphyra species.

Primary mitochondrial disorders and mimics: Insights from a large French cohort.

The objective of this study was to evaluate the implementation of NGS within the French mitochondrial network, MitoDiag, from targeted gene panels to whole exome sequencing (WES) or whole genome sequencing (WGS) focusing on mitochondrial nuclear-encoded genes. Over 2000 patients suspected of Primary Mitochondrial Diseases (PMD) were sequenced by either targeted gene panels, WES or WGS within MitoDiag. We described the clinical, biochemical, and molecular data of 397 genetically confirmed patients, comprising 294 children and 103 adults, carrying pathogenic or likely pathogenic variants in nuclear-encoded genes. The cohort exhibited a large genetic heterogeneity, with the identification of 172 distinct genes and 253 novel variants. Among children, a notable prevalence of pathogenic variants in genes associated with oxidative phosphorylation (OXPHOS) functions and mitochondrial translation was observed. In adults, pathogenic variants were primarily identified in genes linked to mtDNA maintenance. Additionally, a substantial proportion of patients (54% (42/78) and 48% (13/27) in children and adults, respectively), undergoing WES or WGS testing displayed PMD mimics, representing pathologies that clinically resemble mitochondrial diseases. We reported the largest French cohort of patients suspected of PMD with pathogenic variants in nuclear genes. We have emphasized the clinical complexity of PMD and the challenges associated with recognizing and distinguishing them from other pathologies, particularly neuromuscular disorders. We confirmed that WES/WGS, instead of panel approach, was more valuable to identify the genetic basis in patients with "possible" PMD and we provided a genetic testing flowchart to guide physicians in their diagnostic strategy.

Echocardiographic manifestations of mitochondrial disease with GTPBP3 gene mutations: A case report.

Mitochondrial diseases are a group of disorders in which mutations in mitochondrial DNA or nuclear DNA lead to dysfunctional oxidative phosphorylation of cells, with mutations in mitochondrial DNA being the most common cause of mitochondrial disease, and mutations in nuclear genes being rarely reported. The echocardiographic findings of mitochondrial diseases with nuclear gene mutations in children's hearts are even rarer. Even more valuable is that we followed up the patient for 4 years and dynamically observed the cardiac echocardiographic manifestations of mitochondrial disease. Provide ideas for the clinical diagnosis and prognosis of mitochondrial diseases. The patient was seen in the pediatric outpatient clinic for poor strength and mental retardation. echocardiography: mild left ventricular (LV) enlargement and LV wall thickening. Nuclear genetic testing: uanosine triphosphate binding protein 3 (GTPBP3) gene mutation. Diagnosis of mitochondrial disease. Mitochondrial disease with GTPBP3 gene mutations. After receiving drug treatment, the patient exhibited a reduction in lactate levels, an enhanced physical condition compared to prior assessments, and demonstrated average intellectual development. For echocardiographic indications of LV wall thickening and LV enlargement, one needs to be alert to the possibility of hereditary cardiomyopathy, especially in children.

Abstract PS17-09: An FGFR1/2 degrader overcomes antiestrogen resistance in ER+/FGFR1-amplified breast cancer

Abstract We previously reported that nuclear FGFR1 promotes antiestrogen resistance in ER+/FGFR1-amplified breast cancer. Nuclear FGFR1 activity was not affected by FGFR tyrosine kinase inhibitors (TKIs). Furthermore, pan-FGFR TKIs are not well tolerated. Therefore, new therapeutic strategies that inhibit nuclear FGFR1 while sparing FGFR3 and FGFR4 would be required to overcome antiestrogen resistance and limit toxicity. For this study, we used the proteolysis-targeting chimera (PROTAC) DGY-09-192, consisting of the FGFR TKI BCJ398 linked with a VHL recruiting ligand, to degrade FGFR1 and FGFR2. We aimed to determine whether selective degradation of FGFR1/2 overcomes endocrine resistance. Treatment with 50-100 nM DGY-09-192 strongly induced FGFR1 degradation and suppressed phosphorylation of FGFR1 and its downstream targets FRS2, AKT, and ERK1/2 in ER+/FGFR1-amplified CAMA1 and MDA-MB-134 breast cancer cells. FGFR1 degradation was evident within 4 h and lasted 48-96 h after drug washout. The proteasome inhibitor MG132 (10 µM) rescued both FGFR degradation and phosphorylation of downstream signaling molecules induced by DGY-09-192, confirming that FGFR1 degradation is dependent on ubiquitination and proteasomal degradation. At equivalent concentrations, DGY-09-192 suppressed phosphorylation of FGFR1 and its downstream targets more potently than BGJ398. Subcellular fractionation showed treatment with DGY-09-192 (100 nM) degraded FGFR1 in membrane, nuclear, and chromatin-bound fractions in both CAMA1 and MDA-MB-134 cells. Treatment of >CAMA1 cells with DGY-09-192 (>100 nM) downregulated nuclear FGFR1 target genes and canonical ERα target genes, such as CCND1, VEGFA, and CDK12 as measured by qRT-PCR whereas treatment with BGJ398 (100 nM) did not. Treatment with DGY-09-192 (500 nM) reversed resistance to the ER degrader fulvestrant in CAMA1 cells as measured by colony formation assays and using the Incucyte Live-Cell Analysis System. Each drug alone showed a modest effect on cell proliferation whereas the combination completed blocked cell growth (p < 0.0001). Finally, treatment of NOD scid gamma (NSG) mice with established ER+/FGFR1-amplified HCI-011 patient-derived xenografts with DGY-09-192 (40 mg/kg daily x2-6) induced FGFR1 degradation and blocked phosphorylation of downstream targets in the tumors. Studies using the combination of DGY-09-192 and fulvestrant in mice bearing established HCI-011 xenografts are in progress. We next investigated the effect of DGY-09-192 on cancer cells harboring FGFR1 and FGFR2 activating mutations. DGY-09-192 treatment induced FGFR2 degradation and blocked downstream signaling in MFE296 endometrial cancer and EFM-19 breast cancer cells that harbor the FGFR2N549K and FGFR2K659E hotspot mutations, respectively. In addition, DGY-09-192 strongly suppressed MFE296 cell proliferation. We are currently investigating whether DGY-09-192 blocks signaling and cell proliferation induced by the N546K and K656E hotspot FGFR1 mutations in ER+ breast cancer cells. Notably, a recent clinical study showed that tumors harboring these FGFR1 activating mutations are refractory to the pan-FGFR TKI pemigatinib. Conclusions: The FGFR1/2 PROTAC DGY-09-192 induced degradation of nuclear and membrane-bound FGFR1 and blocked FGFR1-induced signaling and nuclear activity in ER+/FGFR1-amplified breast cancer cells. The combination of DGY-09-192 and fulvestrant synergistically suppressed proliferation of these cells. Therefore, FGFR1 degradation represents a promising therapeutic strategy to block FGFR1 activity more completely and selectively in ER+ breast cancers harboring activating FGFR1 alterations. Citation Format: Yasuaki Uemoto, Chang-Ching Lin, Alberto Servetto, Bingnan Wang, Dan Ye, Fabiana Napolitano, Hima Milan Patel, Luigi Formisano, M Rosario Chica-Parrado, Emmanuel Bikorimana, Saurabh Mendiratta, Chuo Chen, Ariella Hanker, Carlos Arteaga. An FGFR1/2 degrader overcomes antiestrogen resistance in ER+/FGFR1-amplified breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS17-09.

Phylogenomics of mulberries (Morus, Moraceae) inferred from plastomes and single copy nuclear genes

Mulberries (genus Morus), belonging to the order Rosales, family Moraceae, are important woody plants due to their economic values in sericulture, as well as for nutritional benefits and medicinal values. However, the taxonomy and phylogeny of Morus, especially for the Asian species, remains challenging due to its wide geographical distribution, morphological plasticity, and interspecific hybridization. To better understand the evolutionary history of Morus, we combined plastomes and a large-scale nuclear gene analyses to investigate their phylogenetic relationships. We assembled the plastomes and screened 211 single-copy nuclear genes from 13 Morus species and related taxa. The plastomes of Morus species were relatively conserved in terms of genome size, gene content, synteny, IR boundary and codon usage. Using nuclear data, our results elucidated identical topologies based on coalescent and concatenation methods. The genus Morus was supported as monophyletic, with M. notabilis as the first diverging lineage and the two North American Morus species, M. microphylla and M. rubra, as sister to the other Asian species. In the Asian Morus species, interspecific relationships were completely resolved. However, cyto-nuclear discordances and gene tree-species tree conflicts were detected in the phylogenies of Morus, with multiple evidences supporting hybridization/introgression as the main cause of discordances between nuclear and plastid phylogenies, while gene tree-species tree conflicts were mainly caused by ILS.

Skimming genomes for systematics and DNA barcodes of corals.

Numerous genomic methods developed over the past two decades have enabled the discovery and extraction of orthologous loci to help resolve phylogenetic relationships across various taxa and scales. Genome skimming (or low-coverage genome sequencing) is a promising method to not only extract high-copy loci but also 100s to 1000s of phylogenetically informative nuclear loci (e.g., ultraconserved elements [UCEs] and exons) from contemporary and museum samples. The subphylum Anthozoa, including important ecosystem engineers (e.g., stony corals, black corals, anemones, and octocorals) in the marine environment, is in critical need of phylogenetic resolution and thus might benefit from a genome-skimming approach. We conducted genome skimming on 242 anthozoan corals collected from 1886 to 2022. Using existing target-capture baitsets, we bioinformatically obtained UCEs and exons from the genome-skimming data and incorporated them with data from previously published target-capture studies. The mean number of UCE and exon loci extracted from the genome skimming data was 1837 ± 662 SD for octocorals and 1379 ± 476 SD loci for hexacorals. Phylogenetic relationships were well resolved within each class. A mean of 1422 ± 720 loci was obtained from the historical specimens, with 1253 loci recovered from the oldest specimen collected in 1886. We also obtained partial to whole mitogenomes and nuclear rRNA genes from >95% of samples. Bioinformatically pulling UCEs, exons, mitochondrial genomes, and nuclear rRNA genes from genome skimming data is a viable and low-cost option for phylogenetic studies. This approach can be used to review and support taxonomic revisions and reconstruct evolutionary histories, including historical museum and type specimens.

Expression and functional analysis of Fushi Tarazu transcription factor 1 (FTZ-F1) in the regulation of steroid hormones during the gonad development of Fujian Oyster, Crassostrea angulata

Crassostrea angulata, a major shellfish cultivated in Southern China, has experienced a notable surge in commercial value in recent years. Understanding the molecular mechanisms governing their reproductive processes holds significant implications for advancing aquaculture practices. In this study, we cloned the orphan nuclear receptor gene, Fushi Tarazu transcription factor 1 (FTZ-F1), of C. angulata and investigated its functional role in the gonadal development. The full-length cDNA of FTZ-F1 spans 2357 bp and encodes a protein sequence of 530 amino acids. Notably, the amino acid sequence of FTZ-F1 in C. angulata shares remarkable similarity with its homologues in other species, particularly in the DNA-binding region (>90%) and ligand-binding region (>44%). In C. angulata, the highest expression level of FTZ-F1 was observed in the ovary, exhibiting more than a 200-fold increase during the maturation stage compared to the initiation stage (P < 0.001). Specifically, FTZ-F1 was mainly expressed in the follicular cells surrounding the oocytes of C. angulata. Upon inhibiting FTZ-F1 gene expression in C. angulata through RNA interference (RNAi), a substantial reduction in the expression of genes involved in the synthesis of sex steroids in the gonads, including 3β-HSD, Cyp17, and follistatin, was observed. In addition, estradiol (E2) and testosterone (T) levels also showed a decrease upon FTZ-F1 silencing, resulting in a delayed gonadal development. These results indicate that FTZ-F1 acts as a steroidogenic factor, participating in the synthesis and regulation of steroid hormones and thus playing an important role in the reproductive and endocrine systems within oysters.

Functional analysis of nuclear receptor genes in molting and metamorphosis of the cigarette beetle, Lasioderma serricorne

Nuclear receptors (NRs) are ligand-regulated transcription factors that are important for the normal growth and development of insects. However, systematic function analysis of NRs in the molting process of Lasioderma serricorne has not been reported. In this study, we identified and characterized 16 NR genes from L. serricorne. Spatiotemporal expression analysis revealed that six NRs were mainly expressed in 3-d-old 4th-instar larvae; five NRs were primarily expressed in 5-d-old adults and four NRs were predominately expressed in prepupae. All the NRs were highly expressed in epidermis, fat body and foregut. RNA interference (RNAi) experiments revealed that knockdown of 15 NRs disrupted the larva–pupa–adult transitions and caused 64.44–100 % mortality. Hematoxylin-eosin staining showed that depletion of 12 NRs prevented the formation of new cuticle and disrupted apolysis of old cuticle. Silencing of LsHR96, LsSVP and LsE78 led to newly formed cuticle that was thinner than the controls. The 20E titer and chitin content significantly decreased by 17.67–95.12 % after 15 NR dsRNA injection and the gene expression levels of 20E synthesis genes and chitin metabolism genes were significantly reduced. These results demonstrated that 15 NR genes are essential for normal molting and metamorphosis of L. serricorne by regulating 20E synthesis and chitin metabolism.

Dietary Black Soldier Fly (Hermetia illucens)-Dipterose-BSF-Enhanced Zebrafish Innate Immunity Gene Expression and Resistance to Edwardsiella tarda Infection.

Dietary management using immunostimulants to protect fish health and prevent bacterial infection is widely practiced. Many insect species possess various bioactive substances that can improve animal health. We previously identified several bioactive polysaccharides derived from insects, including dipterose-BSF from black soldier fly (Hermetia illucens) larvae; this can stimulate innate immunity in mammalian macrophage RAW264.7 cells. However, the effect of dietary dipterose-BSF on the immune system of teleosts remains unclear. Here, we analyzed the immune status of zebrafish (Danio rerio) after 14 days of dietary inclusion of dipterose-BSF (0.01, 0.1, and 1 µg/g), followed by an immersion challenge using Edwardsiella tarda. To identify changes in the transcriptional profile induced by dipterose-BSF, we performed RNA-sequencing analyses of the liver and intestine. Differentially expressed genes were investigated, with both organs showing several upregulated genes, dominated by nuclear factor and tumor necrosis factor family genes. Gene Ontology analysis revealed several terms were significantly higher in the experimental group compared with the control group. Challenge tests suggested that dietary dipterose-BSF had some positive effects on disease resistance in fish, but these effects were not pronounced.

Phylogenetic placement and comparative analysis of the mitochondrial genomes of Idiostoloidea (Hemiptera: Heteroptera).

The classification system and the higher level phylogenetic relationships of Pentatomomorpha, the second largest infraorder of Heteroptera (Insecta: Hemiptera), have been debated and remain controversial over decades. In particular, the placement and phylogenetic relationship of Idiostoloidea are not well resolved, which hampers a better understanding of the evolutionary history of Pentatomomorpha. In this study, for the first time, we reported the complete mitochondrial genome for two narrowly distributed families of Idiostoloidea (including Idiostolidae and Henicocoridae), respectively. The length of the mitochondrial genome of Monteithocoris hirsutus and Henicocoris sp. is 16,632 and 16,013 bp, respectively. The content of AT is ranging from 75.15% to 80.48%. The mitogenomic structure of Idiostoloidea is highly conservative and there are no gene arrangements. By using the Bayesian inference, maximum likelihood, and Bayesian site-heterogeneous mixture model, we inferred the phylogenetic relationships within Pentatomomorpha and estimated their divergence times based on concatenated mitogenomes and nuclear ribosomal genes. Our results support the classification system of six superfamilies within Pentatomomorpha and confirm the monophyletic groups of each superfamily, with the following phylogenetic relationships: (Aradoidea + (Pentatomoidea + (Idiostoloidea + (Coreoidea + (Pyrrhocoroidea + Lygaeoidea))))). Furthermore, estimated divergence times revealed that most pentatomomorphan superfamilies and families diverged during the Late Jurassic to Early Cretaceous, which coincides with the explosive radiation of angiosperms.

A novel PLS-DYW type PPR protein OsASL is essential for chloroplast development in rice

Oryza longistaminata (OL), an AA-genome African wild rice which can propagate clonally via rhizome, is an important germplasm for improvement of Asian cultivated rice, however recessive lethal alleles can hitchhike clonal propagation in heterozygous state. Selfing of OL is difficult due to its self-incompatibility, but simple selfing of hybrid progeny between OL and O. sativa is effective to disclose and eliminate recessive lethal alleles. Here, we identified an exhibited albino-lethal phenotype mutant, from an F2 population between OL and O. sativa, named it albino seedling-lethal (asl). The leaves of asl mutant showed abnormal chloroplast development. The albino characteristics of asl were determined to be governed by a set of recessive nuclear genes through genetic analysis. Map-based cloning experiments found that a single nucleotide variation (G to A) was detected in the exon of OsASL in OL, which causes a premature stop codon. OsASL encodes a PLS-type PPR protein with 12 pentratricopeptide repeat domains, and is translocalized to chloroplasts. Complementation and knockout transgenic experiments further confirmed that OsASL is responsible for the albino-lethal phenotype. Loss-of-function OsASL (i.e. osasl) resulted in devoid of intron splicing of chloroplast RNA atpF, ndhA, rpl2 and rps12, and also RNA editing of ndhB, but facilitates the RNA editing of rpl2 in the plastid. Transcriptome sequencing showed that OsASL was mainly involved in chlorophyll synthesis pathway. The expression of Chlorophyll-associated genes were significantly decreased in asl plants, especially PEP (plastid-encoded RNA polymerase)-mediated genes. Our results suggest that OsASL is crucial for RNA editing, RNA splicing of chloroplast RNA group II genes, and plays an essential role in chloroplast development during early leaf development in rice.

Crucial role of SWL1 in chloroplast biogenesis and development in Arabidopsis thaliana.

The disruption of the SWL1 gene leads to a significant down regulation of chloroplast and secondary metabolites gene expression in Arabidopsis thaliana. And finally results in a dysfunction of chloroplast and plant growth. Although the development of the chloroplast has been a consistent focus of research, the corresponding regulatory mechanisms remain unidentified. In this study, the CRISPR/Cas9 system was used to mutate the SWL1 gene, resulting in albino cotyledons and variegated true leaf phenotype. Confocal microscopy and western blot of chloroplast protein fractions revealed that SWL1 localized in the chloroplast stroma. Electron microscopy indicated chloroplasts in the cotyledons of swl1 lack well-defined grana and internal membrane structures, and similar structures have been detected in the albino region of variegated true leaves. Transcriptome analysis revealed that down regulation of chloroplast and nuclear gene expression related to chloroplast, including light harvesting complexes, porphyrin, chlorophyll metabolism and carbon metabolism in the swl1 compared to wild-type plant. In addition, proteomic analysis combined with western blot analysis, showed that a significant decrease in chloroplast proteins of swl1. Furthermore, the expression of genes associated with secondary metabolites and growth hormones was also reduced, which may be attributed to SWL1 associated with absorption and fixation of inorganic carbon during chloroplast development. Together, the above findings provide valuable information to elucidate the exact function of SWL1 in chloroplast biogenesis and development.

A new method for creating androgenetic diploid fish from tetraploid carp sperm and their genetic composition analysis

The application of cold shock to inhibit the extrusion of the second polar body of fertilized eggs is one of the main methods to induce triploid in diploid fish. In this study, we originally planned to induce hexaploid by this method in tetraploid carp. However, among the surviving offspring, we observed only 50.7% tetraploid and 49.3% diploid individuals, which could potentially result from either androgenetic diploids or gynogenetic diploids. The absence of irradiation and the high proportion of diploids attracted us. However, the lack of sex-specific markers for tetraploid carp, makes it difficult to determine their origin. Fortunately, we also carried out the cross of yellow carp (Cyprinus carpio yellow var., 2n, ♀) × tetraploid carp (♂) and red crucian carp (Carassius auratus red var., 2n, ♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis, diploid offspring have also been found, and whether they have undergone gynogenesis or androgenesis can be judged based on phenotype and genotype. In terms of phenotypes, the key phenotypes of two diploids, such as the whisker length, number of lateral scales, and presence of melanin, are consistent with tetraploid carp. In terms of genotypes, through single-copy nuclear gene cloning and fluorescence in situ hybridization (FISH), it is supported that both diploids are androgenetic offspring. In addition, 14.9% of tetraploid offspring were found in red crucian carp (♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis. By the above-mentioned means of detection, it was found that they were created by inhibiting the extrusion of the second polar body of the fertilized eggs. From this it will be seen that the application of cold shock before the extrusion of the second polar body of fertilized eggs cause not only polyploidy, but also the elimination of both second polar body and female nucleus. Previous studies have found that cold shock can induce lethal androgenetic haploids in diploid fish, but these androgenetic diploid fish produced by tetraploid carp sperm were surviving. We summarize the mechanism behind the above phenomenon, which is mainly related to meiosis occurring at the edge of the cell membrane. Cold shock may disrupt the function of the spindle, resulting in the simultaneous retention or extrusion of female nucleus and second polar body. This study will provide valuable insights into androgenesis, polyploidization, and hybridization in fish genetic breeding.

Validation of a simplified small-scale DNA extraction protocol from wine by quantitative real-time PCR

In the present study, we compared a simplified small-scale purification protocol to obtain DNA admixtures out of wine, with our large-scale published method. The extraction methods must provide DNA free of PCR inhibitors, that can interfere with DNA amplification. To evaluate the efficiency of grapevine’s nuclear DNA extraction from wine, the new protocol was also compared in terms of purity and yield to the DNA obtained out of grapevine’s (Vitis vinifera) leaf tissue, using a commercial kit. Two single-copy nuclear genes, nine-cis-epoxy carotenoid dioxygenase 2 (NCED2), and prefoldin subunit 5-like (PS5) were amplified in DNA extracted from wine and grapevine by real-time TaqMan PCR to determine the presence of inhibitors in relation to the diversity of starting biological matrix. This study showed that the small-scale, simpler method for extracting DNA from wine produced effective results in terms of inhibitor presence and purity. Furthermore, even though the initial biological matrix was more complicated, the grapevine nuclear DNA that was removed from wine was qualitatively equivalent to the DNA that was isolated from the leaves.

Molecular systematics of the subgenus Gephyromantis (Phylacomantis) with description of a new subspecies

The mantellid subgenus Phylacomantis (genus Gephyromantis) currently contains four species of frogs distributed in the South (G. corvus, G. kintana), West (G. atsingy), North East, North, and Sambirano regions (G. pseudoasper) in Madagascar. We assess the molecular systematics of these amphibians based on extended sampling and analysis of multiple nuclear-encoded and mitochondrial genes. We confirm the delimitation of the four known species, which form monophyletic groups in a mitochondrial tree and separate phylogroups without haplotype sharing in haplotype networks of four nuclear markers. Newly included samples of G. pseudoasper from the North and North East regions confirm a comparatively low genetic variation of this species across its range, with pairwise genetic distance in the 16S gene not exceeding 1.4%. On the contrary, newly collected specimens from three sites in the North East and North West of the island strongly differed from all other Phylacomantis, with genetic distances exceeding 4.8% for the16S gene. Mitochondrial and nuclear markers strongly suggest them to be sister to G. corvus from the South. Due to haplotype sharing in two nuclear genes, absence of detectable morphological differences, and lack of data on bioacoustic differentiation, we describe these populations as a new subspecies, G. corvus bakilana ssp. nov., emphasizing that this taxon may be elevated to species status in the future pending new data. This biogeographic pattern, with apparently disjunct sister lineages in the South vs. North West of Madagascar, is rare among Malagasy anurans and calls for increased exploration of the remaining humid forest fragments in the North West and West of the island.

Protective effects of oyster polypeptide on cyclophosphamide-induced immunosuppressed rats.

Oyster polypeptide (OP) is a mixture of oligopeptides extracted from oysters through enzyme lysis, separation, and purification. It is associated with immunomodulatory effects, but the underlying mechanisms are not known. This study therefore combined proton nuclear magnetic resonance (1H-NMR) urinary metabolomics and 16S rRNA gene sequencing of the gut microbiome to determine the immunoprotective mechanisms of OP in rats subjected to cyclophosphamide-induced immunosuppression. Oyster polypeptide restored the body weight and the structure of spleen and thymus in rats with cyclophosphamide-induced immunosuppression. It upregulated the levels of white blood cells (WBCs), hemoglobin (HGB), platelets (PLT), red blood cells (RBCs), immunoglobulin G (IgG), immunoglobulin M (IgM), cytokines such as interleukin‑6 (IL-6) and tumor necrosis factor-α (TNF-α), and increased the numbers of CD3+ and CD4+ T cells in the immunosuppressed rats. The 1H-NMR metabolomics results showed that OP significantly reversed the levels of ten metabolites in urine, including 2-oxoglutarate, citrate, dimethylamine, taurine, N-phenylacetylglycine, alanine, betaine, creatinine, uracil, and benzoate. The 16S rRNA gene sequencing results showed that OP restored the gut microbiome homeostasis by increasing the abundance of beneficial bacteria and reducing the abundance of pathogenic bacteria. Finally, a combination of metabolomics and microbiomics found that the metabolism of taurine and hypotaurine, and the metabolism of alanine, aspartate, and glutamate were disturbed, but these metabolic pathways were restored by OP. This study demonstrated that OP had immunoprotective effects in rats with cyclophosphamide-induced immunosuppression by restoring key metabolic pathways and the gut microbiome homeostasis. Our findings provide a framework for further research into the immunoregulatory mechanisms of OP and its potential use in drugs and nutritional supplements. © 2024 Society of Chemical Industry.

Phylogeographic Pattern of the Assassin Bug Sycanus bifidus Inferred from Mitochondrial Genomes and Nuclear Genes.

The assassin bug Sycanus bifidus has a wide distribution across southern China. This study explored its distribution and evolution by analyzing mitochondrial and nuclear ribosomal RNA genes, revealing how Pleistocene climate and geological changes shaped its phylogeography. We identified two main clades, A and B, that diverged in the Middle Pleistocene. Hainan Island's populations form a unique group within Clade A, suggesting that the Qiongzhou Strait served as a dispersal corridor during glaciation. Rising sea levels likely separated the Hainan population afterward. Ecological niche modeling showed that both populations have been viable since the last interglacial period, with demographic analyses indicating possible expansions during the Middle and Late Pleistocene, driven by favorable climates. This study highlights the significant effects of Pleistocene sea-level and climatic changes on the distribution and evolution of S. bifidus in China.

Protein insertion into the inner membrane of mitochondria: routes and mechanisms.

The inner membrane of mitochondria contains hundreds of different integral membrane proteins. These proteins transport molecules into and out of the matrix, they carry out multifold catalytic reactions and they promote the biogenesis or degradation of mitochondrial constituents. Most inner membrane proteins are encoded by nuclear genes and synthesized in the cytosol from where they are imported into mitochondria by translocases in the outer and inner membrane. Three different import routes direct proteins into the inner membrane and allow them to acquire their appropriate membrane topology. First, mitochondrial import intermediates can be arrested at the level of the TIM23 inner membrane translocase by a stop-transfer sequence to reach the inner membrane by lateral insertion. Second, proteins can be fully translocated through the TIM23 complex into the matrix from where they insert into the inner membrane in an export-like reaction. Carriers and other polytopic membrane proteins embark on a third insertion pathway: these hydrophobic proteins employ the specialized TIM22 translocase to insert from the intermembrane space (IMS) into the inner membrane. This review article describes these three targeting routes and provides an overview of the machinery that promotes the topogenesis of mitochondrial inner membrane proteins.

P155 Gene expression in hypermobility and the role of inflammation

Abstract Background/Aims Hypermobile conditions, including Ehlers-Danlos syndrome and joint hypermobility syndrome, encompass a broad spectrum of heterogenous clinical entities characterised by excessive range of joint movements and variable extra-articular features. The etiological basis of these conditions is thought to relate to abnormal collagen assembly, which impairs the structural integrity of connective tissue, although precise mechanisms remain both debated and incompletely understood. Despite the established strong heritability of hypermobility, a definitive genetic cause remains unidentifiable in most hypermobile patients, highlighting the need to further characterize the genetic landscape of hypermobile conditions. In this study, we therefore employ transcriptomic analysis as an alternate means of elucidating the genetic basis of hypermobility. Methods We tested for the effects of hypermobility on gene expression, at baseline and following an inflammatory challenge, by re-analysing existing transcriptomic data gathered from healthy subjects and patients with fibromyalgia and chronic fatigue using total RNA sequencing of blood samples (Illumina NextSeq 500). From each participant, two blood samples were taken on separate occasions: one after a saline injection and the other after an inflammatory challenge, the typhoid vaccination, in a cross-over design. Participants were assigned to hypermobile or non-hypermobile conditions in accordance with criterion 1: generalized joint hypermobility of the Diagnostic Criteria for Hypermobile Ehlers-Danlos Syndrome (hEDS) produced by the International Consortium on Ehlers-Danlos Syndromes &amp; Related disorders. Differential gene expression analysis comparing hypermobile and non-hypermobile participants was performed using EdgeR software in R studio. Baseline differences were determined by comparing post-saline samples between groups. Differential responses to the inflammatory challenge were detected by comparison of post-typhoid samples between groups. Gene ontology on differentially expressed genes was performed using DAVID’s Functional Annotation Clustering tool. Results We found 288 differentially expressed genes post-saline (i.e at baseline) and 1113 post-typhoid at p &amp;lt; 0.05 although none passed false discovery rate. At baseline, differentially expressed genes were significantly enriched for nuclear, endoplasmic reticulum, and microtubular genes (enrichment score = 2.96, 2.02, 1.3 respectively); and post-typhoid, for 24 clusters, the three most significant being synaptic, ion transport, and plasma membrane genes (enrichment score = 11.34, 8.71, and 7.3 respectively). Conclusion These data demonstrate distinct patterns of gene expression, both at baseline and during inflammation, which implicate potential biological pathways in the pathophysiology of hypermobility. These results also contribute to a growing understanding of the genetic basis of hypermobility and may inform future higher-powered studies to validate our findings. Disclosure J. Porter: None. M. Amato: None. K. Themelis: None. H. Critchley: None. S. Newbury: None. K. Davies: None. N. Harrison: None. B. Towler: None. J. Eccles: Grants/research support; J.E. has received funding from Versus Arthritis, National Institute for Health Research and the Fibroduck Foundation.

BMP Stimulation Differentially Affects Phosphorylation and Protein Stability of β-Catenin in Breast Cancer Cell Lines.

Increased expression and nuclear translocation of β-CATENIN is frequently observed in breast cancer, and it correlates with poor prognosis. Current treatment strategies targeting β-CATENIN are not as efficient as desired. Therefore, detailed understanding of β-CATENIN regulation is crucial. Bone morphogenetic proteins (BMP) and Wingless/Integrated (WNT) pathway crosstalk is well-studied for many cancer types including colorectal cancer, whereas it is still poorly understood for breast cancer. Analysis of breast cancer patient data revealed that BMP2 and BMP6 were significantly downregulated in tumors. Since mutation frequency in genes enhancing β-CATENIN protein stability is relatively low in breast cancer, we aimed to investigate whether decreased BMP ligand expression could contribute to a high protein level of β-CATENIN in breast cancer cells. We demonstrated that downstream of BMP stimulation, SMAD4 is required to reduce β-CATENIN protein stability through the phosphorylation in MCF7 and T47D cells. Consequently, BMP stimulation reduces β-CATENIN levels and prevents its nuclear translocation and target gene expression in MCF7 cells. Conversely, BMP stimulation has no effect on β-CATENIN phosphorylation or stability in MDA-MB-231 and MDA-MB-468 cells. Likewise, SMAD4 modulation does not alter the response of those cells, indicating that SMAD4 alone is insufficient for BMP-induced β-CATENIN phosphorylation. While our data suggest that considering BMP activity may serve as a prognostic marker for understanding β-CATENIN accumulation risk, further investigation is needed to elucidate the differential responsiveness of breast cancer cell lines.