Coding Sequence Research Articles

Genotype B of deformed wing virus and related recombinant viruses become dominant in European honey bee colonies

The Varroa destructor mite’s transmission of deformed wing virus (DWV) to honey bees is responsible for most winter mortalities of colonies worldwide. Four DWV genotypes (A, B, C and D) and numerous recombinants have been described. The most recent studies have reported the greater prevalence of DWV-B over DWV-A in several countries, including European ones, while C and D genotypes appear rare or extinct. However, no global evaluation of DWV-A and DWV-B distribution was available at the European level to date. In this study, we quantified both DWV genotypes by real-time PCR from pools or individual honey bees and from V. destructor mites sampled in 15 European countries between 2010 and 2017. These data and the sequencing of the viral RNA provide a first insight into DWV diversity, with a clear dominance of DWV-B and recombinants (A/B) in Europe. Chimeric sequencing reads were used to locate the recombinant junctions along the DWV genome. These were not randomly distributed, but mainly clustered in three genomic areas: the 5’UTR, leader peptide and helicase coding sequences. In our study, the DWV recombinant genomes shared at least the VP1-VP3 coding sequences with the DWV-B. Further studies are needed to explore the apicultural context explaining these differences in DWV genotype dominance.

The mutational landscape of ARMC5 in Primary Bilateral Macronodular Adrenal Hyperplasia: an update

BackgroundPrimary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a rare cause of Cushing’s syndrome due to bilateral adrenocortical macronodules. Germline inactivating variants of the tumor suppressor gene ARMC5 are responsible for 20–25% of apparently sporadic PBMAH cases and 80% of familial presentations. ARMC5 screening is now routinely performed for PBMAH patients and families. Based on literature review and own observation, this study aims to give an overview of both published and unpublished ARMC5 genetic alterations and to compile the available evidence to discriminate pathogenic from benign variants.Results146 different germline variants (110 previously published and 36 novel) are identified, including 46% missense substitutions, 45% truncating variants, 3% affecting splice sites, 4% in-frame variants and 2% large deletions. In addition to the germline events, somatic 16p loss-of-heterozygosity and 104 different somatic events are described. The pathogenicity of ARMC5 variants is established on the basis of their frequency in the general population, in silico predictions, familial segregation and tumor DNA sequencing.ConclusionsThis is the first extensive review of ARMC5 pathogenic variants. It shows that they are spread on the whole coding sequence. This is a valuable resource for genetic investigations of PBMAH and will help the interpretation of new missense substitutions that are continuously identified.

Codon usage patterns and genomic variation analysis of chloroplast genomes provides new insights into the evolution of Aroideae

Aroideae is an important subfamily of the Araceae family and contains many plants with medicinal and edible value. It is difficult to identify and classify Aroideae species accurately on the basis of morphology alone because of their polymorphic phenotypic traits. The chloroplast genome (CPG) is useful for studying on plant taxonomy and phylogeny, and the analysis of codon usage bias (CUB) in CPGs provides further insights into the intricate phylogenetic relationships among Aroideae. The results showed that the codon third position of the chloroplast genome coding sequence in Aroideae was rich in A and T, with a GC content of 37.91%. The ENC-plot and PR2-plot revealed that the codon usage bias of Aroideae was influenced by multiple factors, with natural selection as the dominant factor. Thirteen to twenty optimal codons ending in A/T were identified in 61 Aroideae species. Additionally, the comparative analysis of CPGs revealed that two single copy regions and non-coding regions were variable in Aroideae. Eight highly divergent regions (Pi > 0.064) were identified (ndhF, rpl32, ccsA, ndhE, ndhG, ndhF-rpl32, ccsA-ndhD, and ndhE-ndhG) , in which ndhE have the potential to serve as a reliable DNA marker to discriminate chloroplasts in Aroideae subfamily. Furthermore, the maximum likelihood-based phylogenetic trees constructed from complete chloroplast genomes and protein-coding sequences presented similar topologies. Principal component clustering analysis based on relative synonymous codon usage values (RSCUs) revealed that Calla was clearly deviated from Montrichardia and Anubias, and that Alocasia was closer to Colocasieae than to Arisaemateae. These findings suggest that the use of RSCU for clustering analysis could offer new theoretical support for species classification and evolution. Our research could provide a theoretical foundation for the chloroplast genetic engineering, taxonomy, and phylogenetic relationships of Aroideae chloroplasts.

A Maize Calmodulin-like 3 Gene Positively Regulates Drought Tolerance in Maize and Arabidopsis

Drought stress is one of the important abiotic stresses that affects maize production. As an important Ca2+ sensor, calmodulin-like proteins (CMLs) play key roles in plant growth, development, and stress response, but there are a limited number of studies regarding CMLs in response to drought stress. In this study, a Calmodulin-like gene, namely ZmCML3, was isolated from maize (Zea mays L.). The coding sequence (CDS) of ZmCML3 was 474 bp and a protein of 158 aa which contains three EF-hand motifs. ZmCML3 was localized within the nucleus and plasma membrane. The expression of ZmCML3 was induced by polyethylene glycol (PEG) 6000, NaCl, methyl jasmonate (MeJA), and abscisic acid (ABA). Overexpression of ZmCML3 resulted in enhanced drought tolerance in maize through increasing proline (Pro) content and the activity of peroxide (POD) and superoxide dismutase (SOD). Meanwhile, ZmCML3 also positively regulated the expression of drought stress-responsive genes in maize under drought stress treatment. Taken together, ZmCML3 acts as a positive regulator in maize response to drought stress. These results will provide theoretical basis for breeding drought tolerance maize variety.

Enterovirus-like particles encapsidate RNA and exhibit decreased stability due to lack of maturation.

To counteract hand, foot, and mouth disease-causing viruses such as enterovirus A71 and coxsackievirus A6, virus-like particles (VLPs) have emerged as a leading contender for the development of a multivalent vaccine. However, VLPs have shown rapid conversion from a highly immunogenic state to a less immunogenic state and low particle integrity lifetimes compared to inactivated virus vaccines, thus raising concerns about their overall stability. Here, we produce VLPs to investigate capsid stability using cryogenic electron microscopy (cryo-EM), mass spectrometry (MS), biochemical assays, and atomic force microscopy (AFM). In contrast to prior studies and prevailing hypotheses, we show that insect-cell produced enterovirus VLPs include encapsidated RNA fragments with viral protein coding sequences. Our integrated approach reveals that CVA6 VLPs do not undergo viral maturation, in contrast to virions; that they can encapsidate RNA fragments, similarly to virions; and that despite the latter, they are more brittle than virions. Interestingly, this indicates that CVA6 VLP stability is more affected by lack of viral maturation than the presence of RNA. Our study highlights how the development of VLPs as vaccine candidates should encompass probing for unwanted (viral) RNA content and establishing control of their maturation to enhance stability.

Novel De Novo Intronic Variant of SYNGAP1 Associated With the Neurodevelopmental Disorders.

SYNGAP1 encodes a Ras/Rap GTPase-activating protein that is predominantly expressed in the brain with the functional roles in regulating synaptic plasticity, spine morphogenesis, and cognition function. Pathogenic variants in SYNGAP1 have been associated with a spectrum of neurodevelopmental disorders characterized by developmental delays, intellectual disabilities, epilepsy, hypotonia, and the features of autism spectrum disorder. The aim of this study was to identify a novel SYNGAP1 gene variant linked to neurodevelopmental disorders and to evaluate the pathogenicity of the detected variant. A novel de novo intronic variant in SYNGAP1 was identified by Whole exome sequencing (WES) and confirmed by Sanger sequencing. Minigene assays were conducted to assess whether the intronic variant in SYNGAP1 influenced the normal splicing of mRNA. A novel de novo intronic variant in SYNGAP1 (c.3582+2T>G) was indentified with clinical features suggestive of neurodevelopmental related disorders. Minigene splicing analysis demonstrated that this noncanonical splice site variant led to the activation of a cryptic acceptor splice site. Consequently, 101 base pairs of intron 16 were aberrantly retained in the mRNA, leading to a frameshift. This frameshift resulted in the introduction of a premature stop codon (TGA) in the coding sequence and the production of a truncated SYNGAP1 protein, potentially leding to loss of function and subsequent disruption of its biological roles. Our findings highlight the significance of de novo pathogenic SYNGAP1 variants at the intron 16/exon 17 junction in the SYNGAP1-related neurodevelopmental disorders, providing novel insights into the genetic basis and diagnosis of these disabilities.

Draft genome dataset of Streptomyces griseoincarnatus strain R-35 isolated from tidal pool sediments.

The marine isolate, Streptomyces griseoincarnatus strain R-35, was isolated from marine sediments collected from the Glencairn Tidal Pool, Table Mountain National Park, Cape Town, South Africa. The genomic DNA was sequenced using the Ion Torrent GeneStudio™ S5 platform, and the de novo assembly was performed using the SPAdes assembler on the Centre for High Performance Computing (CHPC) Lengau Cluster located at the CSIR, Rosebank, South Africa. The draft genome assembly consisted of 722 contigs totaling 7,625,174 base pairs and a G+C% content of 72.2 mol%. Genome completeness and genome contamination were determined as 99.12% and 0.92%, respectively. Genome annotations performed using the Rapid Annotation with Subsystem Technology (RAST) and the Bacterial and Viral Bioinformatics Resource Centre (BV-BRC) determined the presence of 7996 coding sequences (CDS), 63 transfer RNAs (tRNAs), and six ribosomal RNAs (rRNAs). A total of 2570 hypothetical proteins were assigned, and 5246 proteins were assigned to function. The phylogenomic positioning of S. griseoincarnatus strain R-35 was determined using the Type Strain Genome Server (TYGS) and was found to be related to S. griseoincarnatus JCM 4381T, with a digital DNA-DNA hybridisation (dDDH) value of 84.1%, and an OrthoANIu value of 98.22%. The CARD RGI algorithm on Proksee predicted the presence of 6,107 antimicrobial resistance (AMR) features, 27 biosynthetic gene clusters (BGCs) were predicted using antiSMASH, while 189 carbohydrate-active enzymes (CAZymes) were predicted using dbCAN3. The raw genome sequencing data has been submitted to the National Center for Biotechnology (NCBI) under the BioProject ID PRJNA1129156 (BioSample ID Accession Number: SAMN42145163; Short Read Archive (SRA) Accession: SRR29633055; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1129156).

Synthetic alleles to study MUTE-dependent molecular transitions in stomatal development.

Stomatal abundance sets plants' potential for gas exchange, impacting photosynthesis and transpiration and, thus, plant survival and growth. Stomata originate from cell lineages initiated by asymmetric divisions of protodermal cells, producing meristemoids that develop into guard cell pairs. The transcription factors SPEECHLESS, MUTE, and FAMA are essential for stomatal lineage development, sequentially driving cell division and differentiation events. Their absence produces stomataless epidermis, hindering analysis of their roles during lineage development. MUTE drives the transition from proliferating meristemoids to guard mother cells, committed to stomatal fate. We aim to explore the molecular mechanisms underlying MUTE activity, using partial loss-of-function alleles predicted to impair DNA-binding and to potentially alter MUTE transcriptional activity. We engineered mutant allele coding sequences, generated Arabidopsis lines carrying them and analyzed their epidermal and transcriptional phenotypes using microscopy and RNA-seq. Synthetic alleles driven by the MUTE promoter rescued the stomata less phenotype of the seedling-lethal mute-3 mutant, enabling stomata differentiation and resulting in viable, fertile plants. Further examination of the developmental consequences of MUTE partial loss-of-function revealed arrested lineages, reduced stomatal abundance and altered stomatal spacing. Transcriptomic analysis of very young cotyledons from complemented lines indicated that only some MUTE targets require an intact MUTE bHLH domain. Comparison with existing lineage cell-specific transcriptional profiles showed that lineage development in the mutant lines was delayed compared to the wild-type but followed similar gene networks. These synthetic alleles provide new insight into MUTE ability to accurately and timely specify stomata formation.

Small Interference RNA Encapsulated in Liposomes: An Effective Strategy for In vitro Inhibition of SARS-CoV-2 Load

Background: The pressing need for effective SARS-CoV-2 antiviral medicines has driven research into innovative therapeutic techniques. RNA interference with small interfering RNAs (siRNAs) has shown promise as an antiviral treatment. Objective: We evaluated the effectiveness of lipid-based nanoparticles as a viable delivery platform for siRNA-based approach against SARS-CoV-2 in vitro infection. Methods: Liposomes were fabricated by microfluidics to incorporate SARS-CoV-2-specific siRNAs based on conserved sections of the Spike protein coding sequence. Nanoparticle tracking analysis was used to evaluate the nanoparticles' physicochemical features. VERO cell lines infected with SARS-CoV-2 were used to test the efficiency of siRNA-loaded liposomes. RT-PCR was used to determine the viral load by quantifying the SARS-CoV-2 genome. Results: The results showed that liposomes efficiently decreased viral load in infected cells with good physicochemical features, such as a mean particle size of about 180 nm, zeta potential of +2.5 mV and encapsulation efficiency (53.6%). Conclusion: These findings imply that lipid-based nanoparticles might be a targeted delivery strategy for siRNA-based approaches.

Host 3' flap endonuclease Mus81 plays a critical role in trimming the terminal redundancy of hepatitis B virus relaxed circular DNA during covalently closed circular DNA formation.

Hepatitis B virus (HBV) relaxed circular DNA (rcDNA) possesses an 8-9 nucleotide-long terminal redundancy (TR, or r) on the negative (-) strand DNA derived from the reverse transcription of viral pregenomic RNA (pgRNA). It remains unclear whether the TR forms a 5' or 3' flap structure on HBV rcDNA and which TR copy is removed during covalently closed circular DNA (cccDNA) formation. To address these questions, a mutant HBV cell line HepDES-C1822G was established with a C1822G mutation in the pgRNA coding sequence, altering the sequence of 3' TR of (-) strand DNA while the 5' TR remained wild type (wt). The production of HBV rcDNA and cccDNA in HepDES-C1822G cells was comparable to wt levels. Next-generation sequencing (NGS) analysis revealed that the positive (+) strand DNA of rcDNA and both strands of cccDNA predominantly carried the wt nt1822 residue, indicating that the 5' TR of (-) strand DNA serves as the template during rcDNA replication, forming a duplex with the (+) strand DNA, while the 3' TR forms a flap-like structure, which is subsequently removed during cccDNA formation. In a survey of known cellular flap endonucleases using a loss-of-function study, we found that the 3' flap endonuclease Mus81 plays a critical role in cccDNA formation in wild-type HBV replicating cells, alongside the 5' flap endonuclease FEN1. Additionally, we have mapped the potential Mus81 and FEN1 cleavage sites within the TR of nuclear DP-rcDNA by RACE-NGS analyses. The overlapping function between Mus81 and FEN1 in cccDNA formation indicates that the putative 5' and 3' flap formed by TR are dynamically interchangeable on rcDNA precursor. These findings shed light on HBV rcDNA structure and cccDNA formation mechanisms, contributing to our understanding of HBV replication cycle.

Exploring Coding Sequence Length Distributions Across Taxonomic Kingdoms Based on Maximum Information Principle

Background: Genetic information about organisms' traits is stored and encoded in deoxyribonucleic acid (DNA) sequences. The fundamental inquiry into the storage mechanisms of this genetic information within genomes has long been of interest to geneticists and biophysicists. Objective: The objective of this study was to investigate the distribution of coding sequence (CDS) lengths in species genomes across different kingdoms. Methods: In this study, we used the maximum entropy principle and the gamma distribution model based on a comprehensive dataset including viruses, archaea, bacteria, and eukaryote species Results: Our study result revealed unique patterns in CDS length distributions among kingdoms and CDS lengths exhibit a right-skewed distribution, with varying preferences among kingdoms. Eukaryotes displayed bimodal distributions, with CDS sequences longer than those of prokaryotes. Fitting the gamma distribution model revealed differences in shape and scale parameters among kingdoms, with eukaryotes exhibiting larger scale parameters, indicating longer CDS sequences. Additionally, analysis of moments highlighted the complexity of eukaryotic genomes relative to prokaryotes. Conclusion: This study result deepens our understanding of genome evolution and provides valuable insights for biological research.Conclusion: This study result deepens our understanding of genome evolution and provides valuable insights for biological research.

Logical Analysis of Multiple miRNAs with Isothermal Molecular Classifiers Based on LATE-RCA.

Logical analysis of multiple-miRNA expression information and immediate output of diagnostic results facilitates early cancer detection. In this work, we constructed an isothermal molecular classifier capable of performing computations on multiple miRNAs and directly providing diagnosis results. First, we developed linear-after-the-exponential rolling circle amplification (LATE-RCA), a nearly linear isothermal amplification that does not destroy the original quantitative information about miRNAs. By designing different numbers of weighted coding sequences on the circular template, we naturally implemented multiplication in the LATE-RCA process. Summation, subtraction, and reporting were then carried out by strand displacement reactions. The entire workflow of the classifier was validated using synthetic gastric cancer and healthy miRNA samples with an accuracy of 100%, demonstrating its robustness and accuracy. Compared with existing molecular classifiers, our approach performs under isothermal conditions, streamlines computational procedures, and simplifies probe design. We believe that this isothermal molecular classifier has promising prospects in personalized precision medicine.

Natural variation in CTF1 conferring cold tolerance at the flowering stage in rice.

Improving cold tolerance at the flowering stage (CTF) in rice is crucial for minimising yield loss, making the identification and application of cold-tolerant genes and QTLs imperative for effective molecular breeding. The long lead time, dependence on cold treatment conditions, and the inherent complexity of the trait make studying the genetic basis of CTF in rice challenging. To date, the fine-mapping or cloning of QTLs specific to CTF has not yet been achieved. In this study, single segment substitution lines (SSSLs) were constructed using HJX74 as the recipient and IR58025B, known for good CTF, as the donor. This approach led to the identification of two cold tolerance QTLs, qCTF3 and qCTF6, in rice. qCTF6 has promising breeding potential. Further, we identified the causal gene CTF1 underlying qCTF6 through map-based cloning. CTF1 which encodes a conserved putative protein, has two SNPs within its coding sequence that influence CTF in rice. Additionally, genetic variations in the promoter of CTF1 also contributes to CTF. Thirteen variant sites of CTF1 in the four cold tolerance SSSLs are consistent with the IR58025B. Moreover, we analysed 307 accessions to characterise haplotypes based on the 13 variation sites, identifying five distinct haplotypes. The selection and evolutionary analysis indicate that the cold-tolerant haplotype of CTF1 is a newly generated mutation that has undergone selection in japonica during domestication. This study not only provides a novel favourable gene for molecular breeding of CTF but also highlights the potential of CTF1 in advancing rice breeding.

New Bacteriophage Pseudomonas Phage Ka2 from a Tributary Stream of Lake Baikal

Pseudomonas aeruginosa, an opportunistic pathogen, causes various biofilm-associated infections like pneumonia, infections in cystic fibrosis patients, and urinary tract and burn infections with high morbidity and mortality, as well as low treatment efficacy due to the extremely wide spread of isolates with multidrug resistance. Here, we report the new bacteriophage Pseudomonas phage Ka2 isolated from a tributary stream of Lake Baikal and belonging to the Pbunavirus genus. Transmission electron microscopy resolved that Pseudomonas phage Ka2 has a capsid of 57 ± 9 nm and a contractile and inflexible tail of 115 ± 10 nm in the non-contracted state. The genome consists of 66,310 bp with a GC content of 55% and contains 96 coding sequences. Among them, 52 encode proteins have known functions, and none of them are potentially associated with lysogeny. The bacteriophage lyses 21 of 30 P. aeruginosa clinical isolates and decreases the MIC of amikacin, gentamicin, and cefepime up to 16-fold and the MIC of colistin up to 32-fold. When treating the biofilms with Ka2, the biomass was reduced by twice, and up to a 32-fold decrease in the antibiotics MBC against biofilm-embedded cells was achieved by the combination of Ka2 with cefepime for the PAO1 strain, along with a decrease of up to 16-fold with either amikacin or colistin for clinical isolates. Taken together, these data characterize the new Pseudomonas phage Ka2 as a promising tool for the combined treatment of infections associated with P. aeruginosa biofilms.

Type IV collagen derived non-collagenous domain α6 (IV) NC1 and its derivative fragments inhibit endothelial cell proliferation and attenuates in-vivo chorioallantoic membrane angiogenesis.

Targeting tumor angiogenesis with safe endogenous protein inhibitors is a promising therapeutic approach despite the plethora of the first line of emerging chemotherapeutic drugs. The extracellular matrix network in the blood vessel basement membrane and growth factors released from endothelial and tumor cells promote the neovascularization which supports the tumor growth. Contrastingly, small cleaved cryptic fragments of the C-terminal non collagenous domains of the same basement membrane display antiangiogenic effect. In the present study, full length α6(IV)NC1(Hexastatin) and its three subfragments α6S1(IV)NC1, α6S2(IV)NC1, and α6S3(IV)NC1 were validated for their pro-apoptotic and angio-inhibitory property. In order to construct the coding sequence of hexastatin and its three derivative partial peptide fragments were constructed with our proposed method, where the corresponding exons were amplified from the genomic DNA and then assembled together. Coding sequences were cloned and expressed using pLATE31 vector and recombinant proteins were purified with C-terminal His tag. The endogenous NC protein fragments of collagen IV were evaluated in vitro for their role in cytotoxicity on human umbilical vein endothelial cells (HUVECs). The results showed that the NC1 domain and its fragments inhibited the HUVECs cell proliferation, migration, invasion and induced apoptosis. The neovascularization inhibition was studied in in-vitro, via tube formation assay and in-vivo via the CAM Assay. The results showed that blood vessels and inter capillary network were inhibited in endothelial cells and also, in chick embryo treated with recombinant α6(IV)NC1 and its derivatives, except for α6S1(IV)NC1 and these endogenous protein inhibitors act as bio-therapeutics in inhibition of angiogenesis.

Expression evaluation of exogenous and endogenous alcohol dehydrogenase genes in transgenic Arabidopsis.

A lot of endogenous genes, as well as genes from related species, are transformed back into crops for overexpression to improve their corresponding traits. However, almost all of these transgenic events remain at the testing stage. Most of the singular transgenic events of crops approved for commercial release are developed by the transformation and heterologous expression of exogenous genes from distant species. To detect the differences in expression, protein accumulation, and enzyme activity between transformed exogenous and endogenous genes, the coding sequences (CDSs) of the alcohol dehydrogenase (ADH) genes were cloned from dicotyledonous Arabidopsis, monocotyledonous maize, and prokaryotic Escherichia coli, constructed into expression vector pBI121-cMycNY, and used to transform wild-type Arabidopsis, respectively. Three homozygous T3 lines with a single integration site were screened for each of the three transformed genes by antibiotic screening, polymerase chain reaction (PCR) identification, and genomic DNA resequencing. Real-time quantitative PCR (RT-qPCR) analysis showed that the relative expression levels of the transformed exogenous ZmADH and EcADH genes were ten or tens of times higher than that of the transformed endogenous AtADH gene. After confirming the encoded proteins of these transformed genes by Western blotting, enzyme-linked immunosorbent assay (ELISA) showed that the accumulation levels of the proteins encoded by the transformed genes ZmADH and EcADH were significantly higher than that encoded by the transformed endogenous gene AtADH. Enzyme reaction assay showed that the ADH activities of the T3 lines transformed by the exogenous genes ZmADH and EcADH were also significantly higher than that transformed by the endogenous gene AtADH as well as the wild-type control. These results indicated that exogenous genes were more conducive to transgenic improvement of crops.

Isolation, characterization and therapeutic efficacy of lytic bacteriophage ZK22 against Salmonella Typhimurium in mice

BackgroundSalmonella enterica serovar Typhimurium is one of the most common serovars of Salmonella associated with clinical cases. It not only leads to diarrhea and mortality raised in livestock and poultry farming, but also poses a risk to food safety.ResultsIn this study, a lytic bacteriophage named ZK22 was isolated and identified from sewage. It exhibited favorable capability against 20 strains of S. Typhimurium. The genome of ZK22 consisted of a double-stranded DNA with a total length of 47,066 base pairs and a GC content of 45.71%. A total of 78 coding sequences were predicted, with no virulence genes or drug resistance genes predicted. Based on blastn similarity analysis, ZK22 belongs to the genus Skatevirus of the class Caudoviricetes, as a long-tailed Siphovirus. Biological characteristics of ZK22 indicated that the optimal multiplicity of infection (MOI) of bacteriophage ZK22 to S. Typhimurium was 10− 2 (PFU/cell), with an incubation period of around 10 min and the burst size of 393 PFU/cell. The physicochemical resistance results of ZK22 demonstrated that it maintained stability under temperatures ranging from 4 °C to 70 °C and under pH conditions ranging from 3 to 12. After inoculating S. Typhimurium at 37 °C, co-culture mixed with the optimal multiplicity of infection exhibited the lowest OD600 within 12 h, demonstrating the exceptional antibacterial effect. Inoculation of phage ZK22 in mice infected with S. Typhimurium was performed to assess its therapeutic efficacy in vivo. The results showed that phage ZK22 at a dose of 108 PFU/mL increased the survival rates of infected mice, effectively suppressed the dissemination and colonization of the host bacteria in the mice, and alleviated the inflammatory response caused by the infection.ConclusionsIn summary, bacteriophage ZK22 presents favorable application prospects as a potential biological agent for the control of Salmonella infections in livestock and poultry farming.

Single-cell and spatial transcriptomics illuminate bat immunity and barrier tissue evolution.

Bats have adapted to pathogens through diverse mechanisms, including increased resistance - rapid pathogen elimination, and tolerance - limiting tissue damage following infection. In the Egyptian fruit bat (an important model in comparative immunology) several mechanisms conferring disease tolerance were discovered, but mechanisms underpinning resistance remain poorly understood. Previous studies on other species suggested that elevated basal expression of innate immune genes may lead to increased resistance to infection. Here, we test whether such transcriptional patterns occur in Egyptian fruit bat tissues through single-cell and spatial transcriptomics of gut, lung and blood cells, comparing gene expression between bat, mouse and human. Despite numerous recent loss and expansion events of interferons in the bat genome, interferon expression and induction are remarkably similar to that of mouse. In contrast, central complement system genes are highly and uniquely expressed in key regions in bat lung and gut epithelium, unlike in human and mouse. Interestingly, the unique expression of these genes in the bat gut is strongest in the crypt, where developmental expression programs are highly conserved. The complement system genes also evolve rapidly in their coding sequence across the bat lineage. Finally, the bat complement system displays strong hemolytic activity. Together, these results indicate a distinctive transcriptional divergence of the complement system, which may be linked to bat resistance, and highlight the intricate evolutionary landscape of bat immunity.

Emergence of SARS-CoV-2 subgenomic RNAs that enhance viral fitness and immune evasion.

Coronaviruses express their structural and accessory genes via a set of subgenomic RNAs, whose synthesis is directed by transcription regulatory sequences (TRSs) in the 5' genomic leader and upstream of each body open reading frame. In SARS-CoV-2, the TRS has the consensus AAACGAAC; upon searching for emergence of this motif in the global SARS-CoV-2 sequences, we find that it evolves frequently, especially in the 3' end of the genome. We show well-supported examples upstream of the Spike gene-within the nsp16 coding region of ORF1b-which is expressed during human infection, and upstream of the canonical Envelope gene TRS, both of which have evolved convergently in multiple lineages. The most frequent neo-TRS is within the coding region of the Nucleocapsid gene, and is present in virtually all viruses from the B.1.1 lineage, including the variants of concern Alpha, Gamma, Omicron and descendants thereof. Here, we demonstrate that this TRS leads to the expression of a novel subgenomic mRNA encoding a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production and contributes to viral fitness during infection. We observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level.

Prevalent integration of genomic repetitive and regulatory elements and donor sequences at CRISPR-Cas9-induced breaks

CRISPR-Cas9 genome editing has been extensively applied in both academia and clinical settings, but its genotoxic risks, including large insertions (LgIns), remain poorly studied due to methodological limitations. This study presents the first detailed report of unintended LgIns consistently induced by different Cas9 editing regimes using various types of donors across multiple gene loci. Among these insertions, retrotransposable elements (REs) and host genomic coding and regulatory sequences are prevalent. RE frequencies and 3D genome organization analysis suggest LgIns originate from randomly acquired genomic fragments by DNA repair mechanisms. Additionally, significant unintended full-length and concatemeric double-stranded DNA (dsDNA) donor integrations occur when donor DNA is present. We further demonstrate that phosphorylated dsDNA donors consistently reduce large insertions and deletions by almost two-fold without compromising homology-directed repair (HDR) efficiency. Taken together, our study addresses a ubiquitous and overlooked risk of unintended LgIns in Cas9 editing, contributing valuable insights for the safe use of Cas9 editing tools.