Terminal Region Research Articles

An intronic LINE-1 regulates IFNAR1 expression in human immune cells

BackgroundDespite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed.ResultsWe examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1.ConclusionsOur study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study.

Transfer of mRNA Encoding CEBPA in Lipid Nanoparticles to CEBPA Mutated Leukemia Cell Line KO52 and Primary AML Reduces Cell Growth and Induces Myeloid Differentiation

There are known constitutional mutations associated with bone marrow failure and progression to malignancy, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). For the majority of the defects associated with clonal hematopoiesis and progression to MDS/AML, there are no specific treatments. We sought to determine if messenger RNA (mRNA) transfer can restore expression of the normal proteins, modulate abnormal proliferation and differentiation, and enhance response to chemotherapy and targeted agents. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor involved in myeloid differentiation that is mutated in ~7-20% of patients with AML (Su L et al., Frontier Oncol. 2022). A germline alteration was identified in 4.5% of AML patients with CEBPA mutation (Ho PA et al., Blood 2009). While it was originally believed that biallelic mutations of CEBPA confer favorable prognosis, more recently it was realized that a single basic region leucine zipper (bZIP) mutation in the C terminal region suffices to confer favorable risk, as categorized by ELN2022. There are currently no targeted therapies for mutated CEBPA, and no known specific therapy for patients with constitutional or acquired CEBPA mutations. Although most patients with mutated CEBPA achieve initial CR, there is a moderate relapse rate (30-50%). The objective of this study was to determine if restoration of normal CEBPA mRNA would result in expression of normal CEBPA protein in AML cell lines (e.g.-U937), as well as an AML cell line with mutated CEBPA (KO52), and in primary AML blasts, including those with mutated CEBPA. We tested whether expression of normal CEBPA would lead to phenotypic changes including overcoming differentiation block, induce apoptosis, and eliminate leukemic blasts. There are normally two translation initiation sites, leading to production of the CEBPA-p42 or CEBPA-p30 proteins. U937 has high level expression of normal CEBPA-p42, while the KO52 cell line has two CEBPA mutations, p.E50* and p.L324P, the former leading to decrease in CEBPA-p30. To study the consequences of mRNA transfer, we performed immunofluorescence, multicolor flow cytometry (MFC), and Western blot (WB) analysis for non-transfected control cells, GreenLantern mRNA lipid nanoparticle (LNP) transfected control cells, and cells transfected with two types of CEBPA mRNA LNP (#1 and #2). We observed transfection efficiencies of 90-97% in cell lines and 20-95% in primary cells. Figure 1A shows the mean fluorescence intensity (MFI) of Alexa Fluor® 647-labeled monoclonal antibody to the C-terminus of CEBPA. Wild-type CEBPA-p42 was expressed in U937 and KO52 cell lines at 2-4 times control levels by WB quantitation. With two additions of LNP at 0h and 48h, the induced CEBPA protein maintained stable expression up to 5 days. Immunofluorescence demonstrated staining of CEBPA in the nucleus, co-localizing with nuclear DAPI fluorescence. To evaluate the functional consequences of CEBPA introduction, we analyzed morphology by Wright/Giemsa staining, proliferation by cell counting, apoptosis by Annexin V staining, cell cycle using BrdU labeling assay, and immunophenotype with stem and lineage markers including CD45, CD34, CD38, CD123, CD117, HLA-DR, CD13, CD33, CD16, CD11b, CD64, CD14. Strikingly, the enhanced expression of CEBPA was associated with decrease in proliferation and fraction in S phase, and increase in apoptosis and myeloid differentiation, suggesting induction of differentiation that could have therapeutic implications similar to ATRA in acute promyelocytic leukemia. The myeloid differentiation was evident by MFC showing decreased expression of primitive markers CD34, CD117 and HLA-DR, with increased expression of CD64 (Figure 1B), CD38, and CD11b. With this method we have also transfected primary AML patient blasts including a sample that harbored 4 CEBPA mutations. We observed 95% transfection efficiency and >50% decrease in cell number relative to controls with increased myeloid differentiation. Studies of the combination of CEBPA mRNA LNP transfection with targeted inhibitors to the other mutations present in this sample are in progress. Our findings reveal a novel therapeutic strategy using CEBPA mRNA LNP applicable to inherited or acquired disorders of CEBPA. This approach of restoration of normal mRNA could have potential for therapy of many pre-malignant and malignant disorders.

The anti-digestibility mechanism of soy protein isolate hydrolysate on natural starches with different crystal types

The effects of soy protein isolate hydrolysate (SPIH) on the physicochemical properties and digestive characteristics of three starch types (wheat, potato, and pea) were investigated. Fourier-transform infrared spectroscopy and molecular dynamics simulations showed that hydrogen bonds were the driving force of the interaction between SPIH and starch. Furthermore, the SPIH was predicted to preferentially bind to the terminal region of starch using molecular dynamics simulations. Compared to pure starch, adding 20 % SPIH to wheat starch, potato starch, and pea starch, the content of resistant starch increased by 39.71 %, 125.66 % and 37.83 %, respectively. Both the radial distribution function (RDF) and low field-nuclear magnetic resonance (LF-NMR) showed that SPIH reduced the flow of water molecules in starch, indicating that SPIH competed with starch for water molecules. Multiple characterization experiments and molecular dynamics simulations confirmed that the anti-digestibility mechanism of SPIH on natural starches with different crystal types could be attributed to the interaction between starch and SPIH, which decreased the catalytic efficiency of amylase. This study clarified the anti-digestibility mechanism of SPIH on natural starches, which provides new insights into the production of low-glycemic index foods for the diabetic population.

A novel botybirnavirus with a unique satellite dsRNA causes latent infection in Didymella theifolia isolated from tea plants.

A novel botybirnavirus, infecting the tea plant pathogen Didymella theifolia and tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1), together with an additional double-stranded RNA (dsRNA), was characterized. DtBRV1 comprises two dsRNAs (1 and 2) encapsidated in isometric virions, while dsRNA3 is a satellite. The satellite represents a unique specimen since it contains a duplicated region and has high similarity to the two botybirnavirus dsRNAs, supporting the notion that it most likely originated from a deficient genomic component. The biological characteristics of DtBRV1 were further determined. With their unique molecular traits, DtBRV1 and its related dsRNA expand our understanding of virus diversity, taxonomy, and evolution.

Identification of a Novel Pm65 Allele Conferring a Wide Spectrum of Resistance to Powdery Mildew in Wheat Accession PI 351817.

Powdery mildew is caused by the highly adaptive biotrophic fungus Blumeria graminis f. sp. tritici infecting wheat worldwide. Novel powdery mildew resistance genes are urgently needed that can be used rapidly in wheat cultivar development with minimal disruption of trait advances elsewhere. PI 351817 is a German cultivar exhibiting a wide spectrum of resistance to B. graminis f. sp. tritici isolates collected from different wheat-growing regions of the United States. Evaluation of an F2 population and 237 F2:3 lines derived from OK1059060-2C14 × PI 351817 for responses to B. graminis f. sp. tritici isolate OKS(14)-B-3-1 identified a single dominant gene, designated Pm351817, for powdery mildew resistance in PI 351817. Using bulked segregant analysis (BSA) and simple sequence repeat (SSR) markers, Pm351817 was mapped in the terminal region of the long arm of chromosome 2A. Deep sequencing of the genotyping-by-sequencing libraries of the two parental lines identified a set of single-nucleotide polymorphism (SNP) markers in the 2AL candidate gene region. Those SNP markers was subsequently converted to Kompetitive allele-specific PCR (KASP) markers for genotyping the mapping population. Linkage analysis delimited Pm351817 to a 634-kb interval between Stars-KASP656 (771,207,512 bp) and Stars-KASP662 (771,841,609 bp) on 2AL, based on the Chinese Spring reference sequence IWGSC RefSeq v 2.1. Tests of allelism indicated that Pm351817 is located at the Pm65 locus. Pm351817 shows resistance to all B. graminis f. sp. tritici isolates used in this study and can be used to enhance powdery mildew resistance in the United States. KASP markers flanking Pm351817 can be used to select Pm351817 in wheat breeding programs after further tests for polymorphism.

Abstract 16052: Non-Canonical Mechanisms of BARKct-Mediated GRK2 Inhibition in Heart Failure Rescue

Introduction: Significant GRK2 upregulation during HF results in desensitization of βARs. βARKct is a peptide inhibitor of GRK2 comprised of the last carboxyl terminal region of GRK2, and prevents GRK2 binding and βAR desensitization. βARKct overexpression attenuates HF and improves outcomes in animal models. Following oxidative stress, MAPKs phosphorylate the Ser670 (S670) residue of GRK2, which induces GRK2 binding to Hsp90 and localization to mitochondria, initiating pro-death pathways. As S670 is also found in βARKct, it may prevent endogenous GRK2 accumulation in the mitochondria. Hypothesis: βARKct-mediated cardioprotection in HF is due primarily to mitochondrial GRK2 blockade by βARKct. Methods: We generated cardiac-specific mutant βARKct-S670A transgenic mice harboring a Ser-to-Ala mutation at the S670 residue to prevent Hsp90 binding and allow for endogenous GRK2 to continue to translocate to the mitochondria upon ischemic injury, while retaining βARKct in the cytosol to act on βAR signaling pathways. Additionally, we have generated AAV vectors to deliver WT βARKct, βARKct-S670A, and phosphomimetic βARKct-S670D peptides in mice. We performed in vivo hemodynamic analysis to assess cardiac function in βARKct and βARKct-S670A transgenic mice. Additionally, intracellular cAMP levels in AC16 cardiomyocytes transfected with βARKct or βARKct-S670A, and βARKct-S670D plasmids were quantified in response to increasing doses of isoproterenol. Results: Hemodynamic analysis of the βARKct-S670A mice has demonstrated increased baseline contractility in βARKct-S670A mice compared to NLC mice, indicating comparable cardioprotective effects to βARKct mice lacking the point mutation, which are mediated by canonical pathways. In vitro cAMP quantification revealed AC16s transfected with βARKct, βARKct-S670A, and βARKct-S670D plasmids increased cAMP accumulation in response to increasing doses of isoproterenol compared to control. Conclusions: Studies characterizing the mitochondrial pathways involved in HF rescue following ischemic injury are ongoing. These findings will identify new mechanistic information of GRK2 inhibition for HF, and elucidate a new method for attenuating mitochondrial dysfunction during HF.

In vivo liver function reserve assessments in alcoholic liver disease by scalable photoacoustic imaging

We present a rapid and high-resolution photoacoustic imaging method for evaluating the liver function reserve (LFR). To validate its accuracy, we establish alcoholic liver disease (ALD) models and employ dual-wavelength spectral unmixing to assess oxygen metabolism. An empirical mathematical model fits the photoacoustic signals, obtaining liver metabolism curve and LFR parameters. Liver oxygen metabolism significantly drops in ALD with the emergence of abnormal hepatic lobular structure. ICG half-life remarkably extends from 241 to 568 s in ALD. A significant decline in LFR occurs in terminal region compared to central region, indicated by a 106.9 s delay in ICG half-life, likely due to hepatic artery and vein damage causing hypoxia and inadequate nutrition. Reduced glutathione repairs LFR with a 43% improvement by reducing alcohol-induced oxidative damage. Scalable photoacoustic imaging shows immense potential for assessing LFR in alcoholic-related diseases, providing assistance to early detection and management of liver disease.

Germline-restricted chromosomes of the songbirds.

Germline-restricted chromosomes (GRCs) are present in the genomes of germline cells and absent from somatic cells. A GRC is found in all species of the songbirds (Passeri) and in none of the other bird orders studied to date. This indicates that GRC originated in the common ancestor of the songbirds. The germline-restricted chromosome is permanently absent from somatic cells of the songbird, while female germline cells usually contain two copies of GRC and male ones have one copy. In females, GRCs undergo synapsis and restricted recombination in their terminal regions during meiotic prophase. In males, it is almost always eliminated from spermatocytes. Thus, GRC is inherited almost exclusively through the maternal lineage. The germline-restricted chromosome is a necessary genomic element in the germline cells of songbirds. To date, the GRC genetic composition has been studied in four species only. Some GRC genes are actively expressed in female and male gonads, controlling the development of germline cells and synthesis of the proteins involved in the organization of meiotic chromosomes. Songbird species vary in GRC size and genetic composition. The GRC of each bird species consists of amplified and modified copies of genes from the basic genome of that species. The level of homology between GRCs of different species is relatively low, indicating a high rate of genetic evolution of this chromosome. Transmission through the maternal lineage and suppression of the recombination contribute significantly to the accelerated evolution of GRCs. One may suggest that the rapid coordinated evolution between the GRC genes and the genes of the basic genome in the songbirds might be responsible for the explosive speciation and adaptive radiation of this most species-rich and diverse infraorder of birds.

Notes on the terminal region enlargement of a stabilizing NMPC design for a multicopter

This work proposes an NMPC scheme for stabilizing multicopter dynamics with semi-globally asymptotic stability guarantees. Recurrent issues in the state of the art, such as the existence of a terminal invariant set and a decreasing terminal cost under a local controller are analyzed. Within this context, the novelty resides in the use of a nonlinear local controller with feedback linearization properties which allows for arbitrary enlargement of the ellipsoidal terminal region with respect to the initial conditions. The framework is completed through a detailed analysis of the NMPC parameters. Comparisons and simulations for a nanodrone system show the benefits of the approach.

Unveiling the role of PUS7-mediated pseudouridylation in host protein interactions specific for the SARS-CoV-2 RNA genome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, engages in complex interactions with host cell proteins throughout its life cycle. While these interactions enable the host to recognize and inhibit viral replication, they also facilitate essential viral processes such as transcription, translation, and replication. Many aspects of these virus-host interactions remain poorly understood. Here, we employed the catRAPID algorithm and utilized the RNA-protein interaction detection coupled with mass spectrometry technology to predict and validate the host proteins that specifically bind to the highly structured 5' and 3' terminal regions of the SARS-CoV-2 RNA. Among the interactions identified, we prioritized pseudouridine synthase PUS7, which binds to both ends of the viral RNA. Using nanopore direct RNA sequencing, we discovered that the viral RNA undergoes extensive post-transcriptional modifications. Modified consensus regions for PUS7 were identified at both terminal regions of the SARS-CoV-2 RNA, including one in the viral transcription regulatory sequence leader. Collectively, our findings offer insights into host protein interactions with the SARS-CoV-2UTRs and highlight the likely significance of pseudouridine synthases and other post-transcriptional modifications in the viral life cycle. This new knowledge enhances our understanding of virus-host dynamics and could inform the development of targeted therapeutic strategies.

CTG-Net: an efficient cascaded framework driven by terminal guidance mechanism for dilated pancreatic duct segmentation

Pancreatic duct dilation indicates a high risk of various pancreatic diseases. Segmentation for dilated pancreatic duct (DPD) on computed tomography (CT) image shows the potential to assist the early diagnosis, surgical planning and prognosis. Because of the DPD’s tiny size, slender tubular structure and the surrounding distractions, most current researches on DPD segmentation achieve low accuracy and always have segmentation errors on the terminal DPD regions. To address these problems, we propose a cascaded terminal guidance network to efficiently improve the DPD segmentation performance. Firstly, a basic cascaded segmentation architecture is established to get the pancreas and coarse DPD segmentation, a DPD graph structure is build on the coarse DPD segmentation to locate the terminal DPD regions. Then, a terminal anatomy attention module is introduced for jointly learning the local intensity from the CT images, feature cues from the coarse DPD segmentation and global anatomy information from the designed pancreas anatomy-aware maps. Finally, a terminal distraction attention module which explicitly learns the distribution of the terminal distraction regions is proposed to reduce the false positive and false negative predictions. We also propose a new metric called tDice to measure the terminal segmentation accuracy for targets with tubular structures and two other metrics for segmentation error evaluation. We collect our dilated pancreatic duct segmentation dataset with 150 CT scans from patients with five types of pancreatic tumors. Experimental results on our dataset show that our proposed approach boosts DPD segmentation accuracy by nearly 20% compared with the existing results, and achieves more than 9% improvement for the terminal segmentation accuracy compared with the state-of-the-art methods.

Genomic diversity and natural recombination of equid gammaherpesvirus 5 isolates

BackgroundEquid gammaherpesvirus 5 (EHV5) is closely related to equid gammaherpesvirus 2 (EHV2). Detection of EHV5 is frequent in horse populations worldwide, but it is often without a clear and significant clinical impact. Infection in horses can often present as subclinical disease; however, it has been associated with respiratory disease, including equine multinodular pulmonary fibrosis (EMPF). Genetic heterogeneity within small regions of the EHV5 glycoprotein B (gB) sequences have been reported and multiple genotypes of this virus have been identified within individual horses, but full genome sequence data for these viruses is limited. The primary focus of this study was to assess the genomic diversity and natural recombination among EHV5 isolates. ResultsThe genome size of EHV5 prototype strain and the five EHV5 isolates cultured for this study, including four isolates from the same horse, ranged from 181,929 to 183,428 base pairs (bp), with the sizes of terminal repeat regions varying from 0 to 10 bp. The nucleotide sequence identity between the six EHV5 genomes ranged from 95.5 to 99.1%, and the estimated average nucleotide diversity between isolates was 1%. Individual genes displayed varying levels of nucleotide diversity that ranged from 0 to 19%. The analysis of nonsynonymous substitution (Ka > 0.025) revealed high diversity in eight genes. Genome analysis using RDP4 and SplitsTree programs detected evidence of past recombination events between EHV5 isolates. ConclusionGenomic diversity and recombination hotspots were identified among EHV5 strains. Recombination can drive genetic diversity, particularly in viruses that have a low rate of nucleotide substitutions. Therefore, the results from this study suggest that recombination is an important contributing factor to EHV5 genomic diversity. The findings from this study provide additional insights into the genetic heterogeneity of the EHV5 genome.

Computational Probing of the Folding Mechanism of Human Telomeric G-Quadruplex DNA.

The human telomeric (htel) sequences in the terminal regions of human telomeres form diverse G-quadruplex (GQ) structures. Despite much experimental efforts to elucidate the folding pathways of htel GQ, no comprehensive model of htel GQ folding has been presented. Here, we describe folding pathways of the htel GQ determined by state-of-the-art enhanced sampling molecular dynamics simulation at the all-atom level. Briefly, GQ folding is initiated by the formation of a single-hairpin and then followed by the formation of double-hairpins, which then branch via distinct folding pathways to produce different GQ topologies (antiparallel chair, antiparallel basket, hybrids 1 and 2, and parallel propeller). In addition to these double-hairpin states, three-triad and two-tetrad structures in antiparallel backbone alignment serve as key intermediates that connect the GQ folding and transition between two different GQs.

A magnetic fabric study of the origin of englacial debris bands at Fláajökull, Southeast Iceland

Two debris bands associated with an overdeepened basin in the terminus region of the Fláajökull glacier, Iceland, have been sampled for their ice and debris. Here the kinematics of deformation are investigated through anisotropy of magnetic susceptibly (AMS), microstructural analyses, and c-axis preferred orientations. Comparison of the crystallographic orientations of 180 grains and the AMS shape orientations of mineral inclusions in polycrystalline ice reveals a multi-maximum girdle oblique to the planar stratification and a record of near vertical triaxial ellipsoids. These two distinct fabrics can be attributed to both pure and simple shear suggesting a complex deformational history. In addition, AMS from an ice-free sediment ridge on the glacier surface indicates pure shear locally dominated strain. The physical properties of the debris bands, sedimentology and field observations suggest that they were sourced from the glacial bed and emplaced by highly pressurized water into localized fractures.

Cytological origination of the first found allotriploid tree peony, Paeonia × lemoinei ‘Oukan’ (AAB), reveled by molecular karyotype comparation

Polyploid breeding is one of the most important approaches to improve the traits of cultivated plants, but there has been no feasible method to get polyploid tree peony. In this study, we revealed a sexual triploidization approach from a diploid intersubsectional hybrid tree peony, Paeonia × lemoinei ‘High Noon’, to an allotriploid one, P. × lemoinei ‘Oukan’, by accurate karyotyping based on genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). The ancestor genomes from P. suffruticosa and P. delavayi, which were defined as A-genome and B-genome respectively, were clearly distinguished by GISH in ‘High Noon’ and ‘Oukan’. Nonhomologous chromosomes were successfully recognized by means of 5S and 45S rDNA FISH. Karyotype comparation based on GISH and FISH indicated that the B-genome and one of the two A-genomes in ‘Oukan’ (2n=3x=15, AAB) were highly consistent to the corresponding genomes in ‘High Noon’ (2n=2x=10, AB), implying the triploid origination via 2n gametes produced by first division restitution (FDR). In addition, it was demonstrated for the first time that the dual in series sites of 5S rDNA located on the short arm of chromosome 3 in both A and B-genomes. Besides the familiar strong 45S rDNA signals observed at terminal region of short arms, single weak 45S rDNA signal was newly detected at terminal region in the short arm of certain chromosome 1 in A-genome, and at interstitial-distal region in the long arm of chromosome 5 in both genomes. These findings provide a proof and theoretical support for tree peony polyploid breeding, and an accurate karyotyping method for genetic research of plants in Paeonia.

Knee extension tracking and fatigue regulation results using a robust MPC approach in a hybrid exoskeleton

A nonlinear model predictive controller (NMPC) is a promising approach for cooperative control of functional electrical stimulation (FES) and an exoskeleton to assist limb function in people with spinal cord injury (SCI). Potentially, the cooperative control can mitigate the effects of FES-induced muscle fatigue, a significant technical challenge impeding the clinical translation of FES. However, previous NMPC-based cooperative designs have been limited to limb regulation and may not include fatigue regulation in the control objective. In this paper, we design a new NMPC law for limb tracking and fatigue regulation and present experimental results from participants with no disability and a participant with SCI. For robust tracking, this paper augments the NMPC with a Lyapunov-based feedback controller. A terminal controller and terminal constraint regions are specifically derived to show the robust NMPC scheme’s recursive feasibility and to satisfy the input constraints. The experimental results of continuous knee tracking show dynamic allocation of exoskeleton assistance despite decreasing control effectiveness of FES due to muscle fatigue. The robust tube-based MPC reduced the knee angle’s root mean square tracking error by 34% compared to a nominal MPC. It elicited cooperative behavior when it automatically modulated (increased or decreased) FES and motor inputs for different desired fatigue levels while maintaining desired knee angle tracking performance. The statistical t-test showed that the FES input was significantly reduced in the post-fatigue trial (with the desired fatigue level of 1), and the muscle was significantly less fatigued compared to the pre-fatigue experiment (with the desired fatigue level of 0.5).

Karyotype reexamination of Corydoras paleatus (Siluriformes) and a review of the cytogenetics of genus with a focus on the ribosomal genes

The subfamily Corydoradinae is the second largest among the callictids, comprising three genera, with Corydoras Lacépède, 1803 being the largest in the number of species. The genus Corydoras has been divided into five groups of species based on differences in diploid number, chromosomal morphology, and DNA content. Chromosome studies can provide crucial details on population differentiation. To better understand the diversification that occurs in this fish group, the current study based on such markers, presents data from a population of Corydoras paleatus from the first plateau of Paraná, putting them in a comparative scenario. The specimens studied has 2n=44 chromosomes with a karyotypic formula of 18m+26sm. Although conserved, this karyotypic structure shows variation in other populations of C. paleatus already studied, a consequence of chromosomal rearrangements that modify the morphology of chromosomes without modifying the 2n, such as centromeric repositioning. C-banding revealed conspicuous pericentromeric markings in metacentric pairs 4 and 8 and in submetacentric pairs 10 and 14, whose bands have been considered a chromosomal marker not only for C. paleatus but also for the genus. Fluorescent in situ hybridization (FISH) showed the major rDNA (45S) in the terminal region of the long arm of metacentric pair 5. Such location has already been described in other populations of C. paleatus, as they have also been mapped in short arms and with multiple patterns. The present study also provides a review of the genus Corydoras regarding the 2n, karyotypic formula and mainly regarding the number and location of the 45S and 5S rDNA, confirming a scenario in which chromosomal rearrangements have been modeling karyotypes of different populations and settling in the absence of gene flow, a consequence of vicariant events that occurred in the different basins.

The 3' terminal region of Zika virus RNA contains a conserved G-quadruplex and is unfolded by human DDX17.

Zika virus (ZIKV) infection remains a worldwide concern, and currently no effective treatments or vaccines are available. Novel therapeutics are an avenue of interest that could probe viral RNA-human protein communication to stop viral replication. One specific RNA structure, G-quadruplexes (G4s), possess various roles in viruses and all domains of life, including transcription and translation regulation and genome stability, and serves as nucleation points for RNA liquid-liquid phase separation. Previous G4 studies on ZIKV using a quadruplex forming G-rich sequences Mapper located a potential G-quadruplex sequence in the 3' terminal region (TR) and was validated structurally using a 25-mer oligo. It is currently unknown if this structure is conserved and maintained in a large ZIKV RNA transcript and its specific roles in viral replication. Using bioinformatic analysis and biochemical assays, we demonstrate that the ZIKV 3' TR G4 is conserved across all ZIKV isolates and maintains its structure in a 3' TR full-length transcript. We further established the G4 formation using pyridostatin and the BG4 G4-recognizing antibody binding assays. Our study also demonstrates that the human DEAD-box helicases, DDX3X132-607 and DDX17135-555, bind to the 3' TR and that DDX17135-555 unfolds the G4 present in the 3' TR. These findings provide a path forward in potential therapeutic targeting of DDX3X or DDX17's binding to the 3' TR G4 region for novel treatments against ZIKV.

Toll-like receptor may be involved in acquired immune response in pearl oyster Pinctada fucata

The increasing experimental evidence suggests that there are some forms of specific acquired immunity in invertebrates, in which Toll-like receptors (TLRs) play vital roles in activating innate and adaptive immunity and have been comprehensively investigated in mammalian species. Yet, the immune mechanisms underlying TLR mediation in mollusks remain obscure. In this study, we identified a TLR13 gene in the pearl oyster Pinctada fucata for the first time and named it PfTLR13 which consists of a 5′-untranslated terminal region (5′-UTR) of 543 bp, an open reading frame (ORF) of 2667 bp, and a 3′-UTR of 729 bp. We found that PfTLR13 mRNA was expressed in all tissues examined, with the highest level in the gills. The expression of PfTLR13 in the gills of oysters exposed to Vibrio alginolyticus or pathogen-associated molecular patterns (PAMPs) (including LPS, PGN, and poly(I:C)) was significantly higher than in the control group. Interestingly, the immune response to the first stimulation was weaker than the response to the second stimulation, suggesting that the primary stimulation may lead to immune priming of TLR in pearl oysters, similar to acquired immunity in vertebrates. Furthermore, we found that PfTLR13 expression was differentially associated with allograft and xenograft in the pearl oyster P. fucata, with the highest expression levels observed at 12 h post-allograft and 24 h post-xenograft. Overall, our findings provide new insights into the immune mechanisms underlying TLR mediation in mollusks and suggest that PfTLR13 may play a crucial role in the specific acquired immunity of pearl oysters.

Simulations of a protein fold switch reveal crowding-induced population shifts driven by disordered regions

Macromolecular crowding effects on globular proteins, which usually adopt a single stable fold, have been widely studied. However, little is known about crowding effects on fold-switching proteins, which reversibly switch between distinct folds. Here we study the mutationally driven switch between the folds of GA and GB, the two 56-amino acid binding domains of protein G, using a structure-based dual-basin model. We show that, in the absence of crowders, the fold populations PA and PB can be controlled by the strengths of contacts in the two folds, κA and κB. A population balance, PA ≈ PB, is obtained for κB/κA = 0.92. The resulting model protein is subject to crowding at different packing fractions, ϕc. We find that crowding increases the GB population and reduces the GA population, reaching PB/PA ≈ 4 at ϕc = 0.44. We analyze the ϕc-dependence of the crowding-induced GA-to-GB switch using scaled particle theory, which provides a qualitative, but not quantitative, fit of our data, suggesting effects beyond a spherical description of the folds. We show that the terminal regions of the protein chain, which are intrinsically disordered only in GA, play a dominant role in the response of the fold switch to crowding effects.