Large Gene Research Articles

Description and molecular characterisation of Babesia ailuropodae n. sp., a new piroplasmid species infecting giant pandas

BackgroundBabesia spp. are protozoan parasites that infect the red blood cells of domesticated animals, wildlife and humans. A few cases of giant pandas (a flagship species in terms of wildlife conservation) infected with a putative novel Babesia sp. have been reported. However, comprehensive research on the morphological and molecular taxonomic classification of this novel Babesia sp. is still lacking. This study was designed to close this gap and formally describe this new Babesia sp. infecting giant pandas.MethodsDetailed morphological, molecular and phylogenetic analyses were conducted to characterise this Babesia sp. and to assess its systematic relationships with other Babesia spp. Blood samples from giant pandas infected with Babesia were subjected to microscopic examination. The 18S ribosomal RNA (18S rRNA), cytochrome b (cytb) and mitochondrial genome (mitogenome) of the new Babesia sp. were amplified, sequenced and assembled using DNA purified from blood samples taken from infected giant pandas. Based on the newly generated 18S rRNA, cytb and mitogenome sequences, phylogenetic trees were constructed.ResultsMorphologically, the Babesia sp. from giant pandas exhibited various forms, including round to oval ring-shaped morphologies, resembling those found in other small canine Babesia spp. and displaying typical tetrads. Phylogenetic analyses with the 18S rRNA, cytb and mitogenome sequences revealed that the new Babesia sp. forms a monophyletic group, with a close phylogenetic relationship with the Babesia spp. that infect bears (Ursidae), raccoons (Procyonidae) and canids (Canidae). Notably, the mitogenome structure consisted of six ribosomal large subunit-coding genes (LSU1-6) and three protein-coding genes (cytb, cox3 and cox1) arranged linearly.ConclusionsBased on coupled morphological and genetic analyses, we describe a novel species of the genus Babesia, namely, Babesia ailuropodae n. sp., which infects giant pandas.Graphical

Screening for genetic variability in photosynthetic regulation provides insights into salt performance traits in forage sorghum under salt stress

BackgroundSorghum (Sorghum bicolor) is a promising opportunity crop for arid regions of Africa due to its high tolerance to drought and heat stresses. Screening for genetic variability in photosynthetic regulation under salt stress can help to identify target trait combinations essential for sorghum genetic improvement. The primary objective of this study was to identify reliable indicators of photosynthetic performance under salt stress for forage yield within a panel of 18 sorghum varieties from stage 1 (leaf 3) to stage 7 (late flowering to early silage maturity). We dissected the genetic diversity and variability in five stress-sensitive photosynthetic parameters: nonphotochemical chlorophyll fluorescence quenching (NPQ), the electron transport rate (ETR), the maximum potential quantum efficiency of photosystem II (FV/FM), the CO2 assimilation rate (A), and the photosynthetic performance based on absorption (PIABS). Further, we investigated potential genes for target phenotypes using a combined approach of bioinformatics, transcriptional analysis, and homologous overexpression.ResultsThe panel revealed polymorphism, two admixed subpopulations, and significant molecular variability between and within population. During the investigated development stages, the PIABS varied dramatically and consistently amongst varieties. Under higher saline conditions, PIABS also showed a significant positive connection with A and dry matter gain. Because PIABS is a measure of plants’ overall photosynthetic performance, it was applied to predict the salinity performance index (SPI). The SPI correlated positively with dry matter gain, demonstrating that PIABS could be used as a reliable salt stress performance marker for forage sorghum. Eight rubisco large subunit genes were identified in-silico and validated using qPCR with variable expression across the varieties under saline conditions. Overexpression of Rubisco Large Subunit 8 increased PIABS, altered the OJIP, and growth with an insignificant effect on A.ConclusionsThese findings provide insights into strategies for enhancing the photosynthetic performance of sorghum under saline conditions for improved photosynthetic performance and potential dry matter yield. The integration of molecular approaches, guided by the identified genetic variability, holds promise for genetically breeding sorghum tailored to thrive in arid and saline environments, contributing to sustainable agricultural practices.

Split intein-mediated protein trans-splicing to express large dystrophins.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes thatexceed the AAV capacity.

Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus.

Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.

Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method.

Seamless site-directed mutagenesis is an important technique for studying protein functions, tuning enzyme catalytic activities and modifying genetic elements in multiple rounds because it can insert, delete or substitute nucleotides, DNA segments or even entire genes at the target site without introducing any unwanted change. To facilitate seamless site-directed mutagenesis in large plasmids and bacterial artificial chromosomes (BACs) with repetitive sequences, we recently developed the RedEx strategy. Compared with previous methods, our approach achieves the recovery of correct recombinants with high accuracy by circumventing unwanted recombination between repetitive sequences. RedEx readily yields more than 80% accuracy in seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters, which are among the most difficult targets due to the large number of repetitive DNA sequences in modules encoding almost identical enzymes. Here we present the RedEx method by describing in detail the seamless site-directed mutagenesis in a BAC vector. Overall, the process includes three parts: (1) insertion of the RedEx cassette containing the desired mutation together with selection-counterselection markers flanked by unique restriction sites and 20-bp overlapping sequences into the target site by recombineering, (2) removal of the selection-counterselection markers in the BAC by restriction digestion and (3) circularization of the linear BAC by exonuclease-mediated in vitro DNA annealing. This protocol can be performed within 3 weeks and will enable researchers with DNA cloning experience to master seamless site-directed mutagenesis to accelerate their research.

Approach for Phased Sequence-Based Genotyping of the Critical Pharmacogene Dihydropyrimidine Dehydrogenase (DPYD).

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene (DPYD) has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common DPYD variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a cis- or a trans-configuration translates into different toxicity risks and dosing recommendations. DPYD is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of DPYD. We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple DPYD variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism.

Recommendations on approving the name " Entomosporium", with a new species, E.dichotomanthes from China (Leotiomycetes, Drepanopezizaceae).

The phytopathogenic genus, Entomosporium can cause serious leaf diseases worldwide. Entomosporium has long been regarded as a synonym of Diplocarpon. However, different morphologies between Entomosporium and Diplocarpon make this doubtful. Based on morpho-phylogenetic analyses, the placement of the genus was re-evaluated in this study. The combined the internal transcribed spacer gene region (ITS) and the 28S large subunit ribosomal RNA gene region (LSU) phylogenetic analysis shows that Entomosporium is an independent clade within Drepanopezizaceae and formed a sister clade to the generic type Diplocarpon. Moreover, Hymenula and Pseudopeziza do not cluster in Drepanopezizaceae. We propose to resurrect the name Entomosporium, and exclude Hymenulacerealis and Pseudopezizamedicaginis from Drepanopezizaceae and propose to treat them under Ploettnerulaceae. A new species, E.dichotomanthes is also introduced from China based on morpho-molecular analyses which is associated with Dichotomanthestristaniicarpa.

Cerebellar contribution to cognitive deficits and prefrontal cortex dysfunction in Spinocerebellar Ataxia Type 1 (SCA1).

The cerebellum role in cognition and its functional bi-directional connectivity with prefrontal cortex (PFC) is well recognized. However, how chronic cerebellar dysfunction affects PFC function and cognition remains less understood. Spinocerebellar ataxia type 1 (SCA1), is an inherited, fatal neurodegenerative disease caused by an abnormal expansion of glutamine (Q) encoding CAG repeats in the gene Ataxin-1 (ATXN1) and characterized by severe loss of Purkinje cells (PCs) in the cerebellum. Patients with SCA1 suffer from movement and balance deficits, cognitive decline and premature lethality. Cognitive deficits significantly impact patients quality of life, yet how exactly cerebellar degeneration contributes to cognitive deficits and PFC dysfunction in SCA1 is unknown. We have previously demonstrated that expression of mutant ATXN1 only in cerebellar Purkinje cells (PCs) is sufficient to cause cognitive deficits in a transgenic ATXN1[82Q] mouse line. To understand how cerebellar dysfunction impacts the PFC, we examined neuronal activity, synaptic density, and gene expression changes in the PFC of ATXN1[82Q] mice. Remarkably, we found decreased neuronal activity, reduced synaptic density, and altered expression of immediate early genes and pathways involved in glucose metabolism, inflammation and amphetamine in the PFC of ATXN1[82Q] mice. Furthermore, we characterized cellular and molecular PFC dysfunction in a novel conditional knock-in SCA1 line, f-ATXN1146Q mice, expressing floxed human expanded ATXN1 throughout the brain. Intriguingly, we found an increased number of neurons, increased synaptic density and large gene expression alterations in the PFC of f-ATXN1146Q mice. Finally, to precisely determine the role of cerebellar dysfunction in cognitive deficits and PFC dysfunction in SCA1, we crossed f-ATXN1146Q mice with Pcp2-Cre mice expressing Cre recombinase in PCs to delete expanded ATXN1 only in PCs. Surprisingly, we have found that deleting expanded ATXN1 in PCs exacerbated cognitive deficits and PFC dysfunction in these mice. Our findings demonstrate that circumscribed cerebellar dysfunction is sufficient to impact PFC activity and synaptic connectivity impairing cognition. However, when multiple brain regions are impacted in disease, cerebellar dysfunction may ameliorate PFC pathology and cognitive performance.

Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus

The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Quantitative imaging reveals the role of MpARF proteasomal degradation during gemma germination

The auxin signaling molecule controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of the ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and DNA-binding ARF transcription factors. Although extensive qualitative understanding of the pathway and its interactions has been obtained, mostly by studying the flowering plant Arabidopsis thaliana, it remains unknown how these translate to quantitative system behavior in vivo, a problem that is confounded by the large NAP gene families in most species. Here, we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knockin fluorescent fusion proteins. Beyond revealing the dynamic native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs, MpARF1 and MpARF2, are proteasomally degraded. This auxin-independent degradation tunes ARF protein stoichiometry to favor gene activation, thereby reprogramming auxin response during the developmental progression. Thus, quantitative analysis of the entire NAP has enabled us to identify ARF degradation and the stoichiometries of activator and repressor ARFs as a potential mechanism for controlling gemma germination.

GALEON: a comprehensive bioinformatic tool to analyse and visualize gene clusters in complete genomes.

Gene clusters, defined as a set of genes encoding functionally related proteins, are abundant in eukaryotic genomes. Despite the increasing availability of chromosome-level genomes, the comprehensive analysis of gene family evolution remains largely unexplored, particularly for large and highly dynamic gene families or those including very recent family members. These challenges stem from limitations in genome assembly contiguity, particularly in repetitive regions such as large gene clusters. Recent advancements in sequencing technology, such as long reads and chromatin contact mapping, hold promise in addressing these challenges. To facilitate the identification, analysis, and visualization of physically clustered gene family members within chromosome-level genomes, we introduce GALEON, a user-friendly bioinformatic tool. GALEON identifies gene clusters by studying the spatial distribution of pairwise physical distances among gene family members along with the genome-wide gene density. The pipeline also enables the simultaneous analysis and comparison of two gene families and allows the exploration of the relationship between physical and evolutionary distances. This tool offers a novel approach for studying the origin and evolution of gene families. GALEON is freely available from https://www.ub.edu/softevol/galeon and https://github.com/molevol-ub/galeon.

Can genomic signatures guide the selection of host-specific agents for weed biological control?

Biological control of weeds involves deliberate introduction of host-specific natural enemies into invaded range to reduce the negative impacts of invasive species. Assessing the specificity is a crucial step, as introduction of generalist natural enemies into a new territory may pose risks to the recipient communities. A mechanistic understanding of host use can provide valuable insights for the selection of specialist natural enemies, bolster confidence in non-target risk assessment and potentially accelerate the host specificity testing process in biological control. We conducted a comprehensive analysis of studies on the genomics of host specialization with a view to examine if genomic signatures can help predict host specificity in insects. Focusing on phytophagous Lepidoptera, Coleoptera and Diptera, we compared chemosensory receptors and enzymes between "specialist" (insects with narrow host range) and "generalist" (insects with wide host range) insects. The availability of genomic data for biological control agents (natural enemies of weeds) is limited thus our analyses utilized data from pest insects and model organisms for which genomic data are available. Our findings revealed that specialists generally exhibit a lower number of chemosensory receptors and enzymes compared with their generalist counterparts. This pattern was more prominent in Coleoptera and Diptera relative to Lepidoptera. This information can be used to reject agents with large gene repertoires to potentially accelerate the risk assessment process. Similarly, confirming smaller gene repertoires in specialists could further strengthen the risk evaluation. Despite the distinctive signatures between specialists and generalists, challenges such as finite genomic data for biological control agents, ad hoc comparisons, and fewer comparative studies among congeners limit our ability to use genomic signatures to predict host specificity. A few studies have empirically compared phylogenetically closely related species, enhancing the resolution and the predictive power of genomics signatures thus suggesting the need for more targeted studies comparing congeneric specialists and generalists.

Pathogenic variants in KMT2C result in a neurodevelopmental disorder distinct from Kleefstra and Kabuki syndromes

Trithorax-related H3K4 methyltransferases, KMT2C and KMT2D, are critical epigenetic modifiers. Haploinsufficiency of KMT2C was only recently recognized as a cause of neurodevelopmental disorder (NDD), so the clinical and molecular spectrums of the KMT2C-related NDD (now designated as Kleefstra syndrome 2) are largely unknown. We ascertained 98 individuals with rare KMT2C variants, including 75 with protein-truncating variants (PTVs). Notably, ∼15% of KMT2C PTVs were inherited. Although the most highly expressed KMT2C transcript consists of only the last four exons, pathogenic PTVs were found in almost all the exons of this large gene. KMT2C variant interpretation can be challenging due to segmental duplications and clonal hematopoesis-induced artifacts. Using samples from 27 affected individuals, divided into discovery and validation cohorts, we generated a moderate strength disorder-specific KMT2C DNA methylation (DNAm) signature and demonstrate its utility in classifying non-truncating variants. Based on 81 individuals with pathogenic/likely pathogenic variants, we demonstrate that the KMT2C-related NDD is characterized by developmental delay, intellectual disability, behavioral and psychiatric problems, hypotonia, seizures, short stature, and other comorbidities. The facial module of PhenoScore, applied to photographs of 34 affected individuals, reveals that the KMT2C-related facial gestalt is significantly different from the general NDD population. Finally, using PhenoScore and DNAm signatures, we demonstrate that the KMT2C-related NDD is clinically and epigenetically distinct from Kleefstra and Kabuki syndromes. Overall, we define the clinical features, molecular spectrum, and DNAm signature of the KMT2C-related NDD and demonstrate they are distinct from Kleefstra and Kabuki syndromes highlighting the need to rename this condition.

Detection of Klebsiella pneumoniae Carbapenem Resistance Genes by qPCR: Choosing the Right Method for Total DNA Extraction.

Rapid and accurate detection of Klebsiella pneumoniae carbapenem resistance is important for infection control and targeted antibiotic therapy. PCR-based assay performance heavily depends on the quality and quantity of template DNA. Challenges arise from the necessity to isolate chromosomal and large plasmid-encoded resistance genes simultaneously from a limited number of target cells and to remove PCR inhibitors. qPCRs for the detection of K. pneumoniae strains carrying blaOXA-48, blaNDM-1, blaKPC-2, and blaVIM-1 carbapenemase genes were developed. We compared the performance of template DNA extracted with silica column-based methods, reversed elution systems, and lysis-only methods either from diluted culture fluid or from a synthetic stool matrix which contained PCR inhibitors typically present in stool. The synthetic stool matrix was chosen to mimic K. pneumoniae containing rectal swabs or stool samples in a reproducible manner. For total DNA isolated from culture fluid, resistance gene detection by qPCR was always possible, independent of the extraction method. However, when total DNA was isolated from synthetic stool matrix spiked with K. pneumoniae, most methods were insufficient. The best performance of template DNA was obtained with reversed elution. This highlights the importance of choosing the right DNA extraction method for consistent carbapenem resistance detection by PCR.

Two members of a Nodule-specific Cysteine-Rich (NCR) peptide gene cluster are required for differentiation of rhizobia in Medicago truncatula nodules.

Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.

Trans-complementation of the viral movement protein mediates efficient expression of large target genes via a tobacco mosaic virus vector.

The development of plant virus-based expression systems has expanded rapidly owing to their potential applications in gene functional and disease resistance research, and industrial production of pharmaceutical proteins. However, the low yield of certain proteins, especially high-molecular-mass proteins, restricts the production scale. In this study, we observed that the tobacco mosaic virus (TMV)-mediated expression of a foreign protein was correlated with the amount of the movement protein (MP) and developed a TMV-derived pAT-transMP vector system incorporating trans-complementation expression of MP. The system is capable of efficient expression of exogenous proteins, in particular those with a high molecular mass, and enables simultaneous expression of two target molecules. Furthermore, viral expression of competent CRISPR-Cas9 protein and construction of CRISPR-Cas9-mediated gene-editing system in a single pAT-transMP construct was achieved. The results demonstrated a novel role for TMV-MP in enhancing the accumulation of a foreign protein produced from the viral vector or a binary expression system. Further investigation of the mechanism underlying this role will be beneficial for optimization of plant viral vectors with broad applications.

Search for genes gained by horizontal gene transfer in an entomopoxvirus, with special reference to the analysis of the transfer of an ABC transporter gene

Although it is generally believed that large DNA viruses capture genes by horizontal gene transfer (HGT), the detailed manner of such transfer has not been fully elucidated. Here, we searched for genes in the coleopteran entomopoxvirus (EV) Anomala cuprea entomopoxvirus (ACEV) that might have been gained by ACEV by HGT. We classified the potential source organisms for HGT into three categories: the host A. cuprea; other organisms, including viruses unrelated to EVs; and organisms with uncertain host attribution. Of the open reading frames (ORFs) of the ACEV genome, 2.1 % were suggested to have been gained from the host by ACEV or its recent ancestor via HGT; 8.7 % were possibly from organisms other than the host, and 3.7 % were possibly from the third category of organisms via HGT. The analysis showed that ACEV contains some interesting ORFs obtained by HGT, including a large ATP-binding cassette protein (ABC transporter) ORF and a tenascin ORF (IDs ACV025 and ACV123, respectively). We then performed a detailed analysis of the HGT of the ACEV large ABC transporter ORF—the largest of the ACEV ORFs. mRNA sequences obtained by RNA-seq from fat bodies—sites of ACEV replication—and midgut tissues—sites of initial infection—of the virus's host A. cuprea larvae were subjected to BLAST analysis. One type of ABC transporter ORF from the fat bodies and two types from the midgut tissues, one of which was identical to that in the fat bodies, had the greatest identity to the ABC transporter ORF of ACEV. The two types from the host had high levels of identity to each other (approximately 95 % nucleotide sequence identity), strongly suggesting that the host ABC transporter group consisting of the two types was the origin of ACV025. We then determined the sequence (12,381 bp) containing a full-length gene of the A. cuprea ABC transporter. It turned out to be a transcription template for the abovementioned mRNA found in both tissues. In addition, we determined a large part (ca. 6.9 kb) of the template sequence for the mRNA found only in the midgut tissues. The results showed that the ACEV ABC transporter ORF is missing parts corresponding to introns of the host ABC transporter genes, indicating that the ORF was likely acquired by HGT in the form of mRNA. The presence of definite duplicated sequences adjacent to the ACEV ABC transporter genes—a sign of LINE-1 retrotransposon-mediated HGT—was not observed. An approximately 2-month ACV025 transcription experiment suggested that the transporter sequence is presumed to be continuously functional. The amino acid sequence of ACV025 suggests that its product might function in the regulation of phosphatide in the host-cell membranes.

Integrative analyses on the ciliates Colpoda illuminate the life history evolution of soil microorganisms.

Microorganisms play a central role in sustaining soil ecosystems and agriculture, and these functions are usually associated with their complex life history. Yet, the regulation and evolution of life history have remained enigmatic and poorly understood, especially in protozoa, the third most abundant group of organisms in the soil. Here, we explore the life history of a cosmopolitan species-Colpoda steinii. Our analysis has yielded a high-quality macronuclear genome for C. steinii, with size of 155 Mbp and 37,123 protein-coding genes, as well as mean intron length of ~93 bp, longer than most other studied ciliates. Notably, we identify two possible whole-genome duplication events in C. steinii, which may account for its genome being about twice the size of C. inflata's, another co-existing species. We further resolve the gene expression profiles in diverse life stages of C. steinii, which are also corroborated in C. inflata. During the resting cyst stage, genes associated with cell death and vacuole formation are upregulated, and translation-related genes are downregulated. While the translation-related genes are upregulated during the excystment of resting cysts. Reproductive cysts exhibit a significant reduction in cell adhesion. We also demonstrate that most genes expressed in specific life stages are under strong purifying selection. This study offers a deeper understanding of the life history evolution that underpins the extraordinary success and ecological functions of microorganisms in soil ecosystems.IMPORTANCEColpoda species, as a prominent group among the most widely distributed and abundant soil microorganisms, play a crucial role in sustaining soil ecosystems and promoting plant growth. This investigation reveals their exceptional macronuclear genomic features, including significantly large genome size, long introns, and numerous gene duplications. The gene expression profiles and the specific biological functions associated with the transitions between various life stages are also elucidated. The vast majority of genes linked to life stage transitions are subject to strong purifying selection, as inferred from multiple natural strains newly isolated and deeply sequenced. This substantiates the enduring and conservative nature of Colpoda's life history, which has persisted throughout the extensive evolutionary history of these highly successful protozoa in soil. These findings shed light on the evolutionary dynamics of microbial eukaryotes in the ever-fluctuating soil environments. This integrative research represents a significant advancement in understanding the life histories of these understudied single-celled eukaryotes.

Congenital hallux valgus occurs in Fibrodysplasia Ossificans Progressiva and BMPR1B-associated dysplasia: an important distinction

BackgroundFibrodysplasia Ossificans Progressiva (FOP; OMIM #135100) is an ultrarare genetic disorder characterised by congenital bilateral hallux valgus (CBHV), intermittent soft tissue swellings and progressive heterotopic ossification. We report a three-month-old girl with great toe abnormalities similar to FOP, in whom comprehensive clinical workup and genetic investigations illustrates an alternative diagnosis.Case presentationA three-month-old girl presented with CBHV. The antenatal period was unremarkable, she was born by spontaneous vaginal delivery with an uneventful subsequent course, except for maternal concern of her bent toes which received reassurance from several health professionals. Her mother’s persisting concerns were explored via the internet and social media leading her to request referral to an expert bone centre for consideration of FOP. On examination, she was thriving, there was no dysmorphism, subcutaneous lumps, skeletal or extra-skeletal deformity except for shortened great toes with lateral deviation of the proximal and distal phalanges. FOP was a feasible diagnosis, for which CBHV is highlighted as an early sign. A cautionary potential diagnosis of FOP was counselled, including advice to defer intramuscular immunisations until genetic results available. Genetic investigation was undertaken through rapid whole genomic sequencing (WGS), with analysis of data from a skeletal dysplasia gene panel, which demonstrated no ACVR1variants. The only finding was a heterozygous variant of unknown significance in BMPR1B (c1460T>A, p.(Val487Asp)), which encodes a bone morphogenic receptor involved in brachydactyly syndromes A1, A2 and D and acromesomelic dysplasia 3 (only the latter being an autosomal recessive condition).ConclusionThis report highlights that CBHV serves as a vital diagnostic indicator of FOP and affected infants should be considered and investigated for FOP, including precautionary management whilst awaiting genetic studies. The second educational aspect is that CBHV may not represent a generalised skeletal disorder, or one much less significant than FOP. Receptor-ligand BMP and Activins mediated interactions are instrumental in the intricate embryology of the great toe. Recognition of non-FOP conditions caused by alterations in different genes are likely to increase with new genomic technology and large gene panels, enhancing understanding of bone signaling pathways.