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As the most prevalent companion animal, cats are threatened by numerous infectious diseases and carry zoonotic pathogens such as Toxoplasma gondii and Bartonella henselae, which are the primary causes of human toxoplasmosis and cat-scratch disease. Vaccines play a crucial role in preventing and controlling the spread of diseases in both humans and animals. Currently, there are only three core vaccines available to prevent feline panleukopenia, feline herpesvirus, and feline calicivirus infections, with few vaccines available for other significant feline infectious and zoonotic diseases. Feline herpesvirus, a major component of the core vaccine, offers several advantages and a stable genetic manipulation platform, making it an ideal model for vaccine vector development to prevent and control feline infectious diseases. This paper reviews the technologies involved in the research and development of the feline herpesvirus vaccine vector, including homologous recombination, CRISPR/Cas9, and bacterial artificial chromosomes. It also examines the design and effectiveness of expressing antigens of other pathogens using the feline herpesvirus as a vaccine vector. Additionally, the paper analyzes existing technical bottlenecks and challenges, providing an outlook on its application prospects. The aim of this review is to provide a scientific basis for the research and development of feline herpesvirus as a vaccine vector and to offer new ideas for the prevention and control of significant feline infectious and zoonotic diseases. [ABSTRACT FROM AUTHOR]

Our goal with this Microorganisms Special Issue on Marek's disease virus was to collect manuscripts that would provide deeper insights into MDV infection, lytic replication, and latency in vitro and in vivo. Two papers addressed pressing questions in MDV research using CRISPR/Cas9 approaches: the switch between lytic and latent MDV infections and the use of gene technology to generate MDV-resistant chickens. Marek's disease virus (MDV), an Alphaherpesvirus belonging to the genus Mardivirus, causes T cell lymphomas in chickens and remains one of the greatest threats to poultry production worldwide. [Extracted from the article]

Human coronaviruses are a continuous threat to the human population and have limited antiviral treatments, and the recent COVID-19 pandemic sparked interest in finding new antiviral strategies, such as natural products, to combat emerging coronaviruses. Rapid efforts in the scientific community to identify effective antiviral agents for coronaviruses remain a focus to minimize mortalities and global setbacks. In this study, an essential oil derived from Myrtus communis L. (MEO) is effective against HCoV-229E and HCoV-OC43 virus infections in comparison to two FDA-approved drugs, Remdesivir and Nirmatrelvir. Gas-chromatography and mass spectrometry were used to identify the chemical composition of MEO. Slight antioxidant activity was observed in MEO, indicating a role in oxidative stress. A dose–response curve measuring the EC50 indicates a high potency against HCoV-229E and HCoV-OC43 virus infections on Huh7.5 cells with low cytotoxicity using a PrestoBlue cell viability assay. Our findings demonstrate that MEO exhibits potent antiviral activity against HCoV-229E and HCoV-OC43 on Huh7.5 cells within a low-cytotoxicity range, but not on SARS-CoV-2. Artificial bacterial chromosome plasmids that expressed SARS-CoV-2 used for replicon—to determine viral replication and viral assembly/egress on HEK293T/17 cells—and virus-like particles on Huh7.5-AT cells—to determine viral entry and assembly/egress—showed no antiviral activity with MEO in comparison to Remdesivir. This study reveals the potential effectiveness of MEO as an alternative natural remedy to treat human coronaviruses and a potential antiviral agent for future coronavirus infections. [ABSTRACT FROM AUTHOR]

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications. [ABSTRACT FROM AUTHOR]

The aberrant expression of specific long noncoding RNAs (lncRNAs) has been associated with cognitive and psychiatric disorders. Although a growing number of lncRNAs are now known to regulate neural cell development and function, relatively few lncRNAs have been shown to underlie animal behavior. Pnky is an evolutionarily conserved, neural lncRNA that regulates brain development. Using mouse genetic strategies, we show that Pnky has sex-specific roles in mouse behavior and that this lncRNA can underlie specific behavior by functioning in trans. Male Pnky-knockout mice have decreased context generalization in a paradigm of associative fear learning and memory. In female Pnky-knockout mice, there is an increase in the acoustic startle response, a behavior that is altered in affective disorders. Remarkably, expression of Pnky from a bacterial artificial chromosome transgene decreases the acoustic startle response in female Pnky-knockout mice, demonstrating that Pnky can modulate specific animal behavior by functioning in trans. More broadly, these studies illustrate how specific lncRNAs can underlie cognitive and mood disorders. Relatively few lncRNAs have been shown to regulate animal behavior. Here, Saha et al. use mouse genetic methods to show that the lncRNA Pnky can underlie specific mouse behavior by functioning in trans. [ABSTRACT FROM AUTHOR]

The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper, we characterize the EBV present in 21 pediatric and adult patients who were treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) were of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family in which several members have persistent T or NK cell infection by EBV indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient and not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children. IMPORTANCE EBV contributes to several types of human cancer. Some cancers and nonmalignant lymphoproliferative diseases involving T or NK cells contain EBV. These diseases are relatively frequent in Japan and China and have been shown sometimes to have deletions in the EBV genome in the disease cells. We identify further examples of deletions within the EBV genome associated with T or NK cell diseases, and we provide evidence that the virus genomes with these deletions are most likely selected in the individual cases, rather than being transmitted between people during infection. We demonstrate EBV infection of cord blood T cells by highly characterized, cloned EBV genomes and suggest that transient infection of T cells may be part of normal asymptomatic infection by EBV in young children. [ABSTRACT FROM AUTHOR]

The Neotropical deer genus Mazama is characterized by homoplastic morphological characters, a high karyotypic diversity, and a polyphyletic condition. The species of the genus have been recovered into two multigeneric lineages, the subtribes Odocoileina and Blastocerina, of the tribe Odocoileini (New World deer) in the family Cervidae. Within the Blastocerina, gray brockets include two non-sister species, Subulo gouazoubira, occurring south of the Amazon region, and Passalites nemorivagus, occurring in the Guianas and in the Amazon region. We clarify the taxonomic status and phylogenetic position of Mazama americana citus Osgood, 1912 (referred to as either S. gouazoubira or P. nemorivagus by other authors). We collected a topotype of M. a. citus from the eastern shore of Lake Maracaibo, Venezuela, characterize it morphologically and cytogenetically (conventional banding and fluorescence in situ hybridization), and carry out a phylogenetic analysis of its whole mitogenome and Cytb alongside two additional specimens of M. a. citus from northwestern Venezuela. Our analyses reveal the topotype to be a large gray brocket with a cinnamon band above the eyes and 2n = 61 and FN = 70 karyotype. Using cattle whole chromosome painting and bacterial artificial chromosome X probes, we determined its karyotype to differ in at least 10 rearrangements from that of S. gouazoubira. Bayesian inference recovers M. a. citus within the Blastocerina subtribe, separated phylogenetically from other gray brockets (100% branch value), revealing the Osgood's gray brocket to be a valid species that should be assigned to a new genus. We propose the generic name Bisbalus, with Bisbalus citus (Osgood, 1912) as the type species. [ABSTRACT FROM AUTHOR]

In response to Pi deprivation, phosphate 1 (PHO1) is a significant regulator at trans-eQTL hotspots in Brassica rapa. Brassica rapa short-read sequencing data analysis revealed four PHO1 paralog genes, PHO1_A, PHO1_B, PHO1_C, and PHO1_D, placed in tandem with very high sequence similarity. However, based on short-read genomic sequence data, only three transcripts are accessible. Five bacterial artificial chromosomes (BACs) can be sequenced using a long-read sequencer, which improves de novo assembly and identifies structural variants. The PHO1 gene's quadruplicating tandem positions in the genomic sequence were confirmed by an analysis of long-read data. Transcript analysis identified only three groups of PHO1 paralogs (ortholog AT1G14040 in Arabidopsis), i.e., PHO1_A, PHO1_B, and PHO1_D, expressed in B. rapa leaf tissues under Pi deficiency. PHO1_A, with transcript ID XM_009150437.2, has five different splice variants found. These splice variants' truncated proteins demonstrated PHO1_A's function in P control as opposed to protein encoding. [ABSTRACT FROM AUTHOR]

Macroptilium (Benth.) Urb. is a neotropical legume genus from the subtribe Phaseolinae. The investigated species present a stable chromosome number (2n = 22), but differ in their karyotype formulae, suggesting the presence of chromosome rearrangements. In this work, we comparatively analysed the karyotypes of six species (Macroptilium atropurpureum, Macroptilium bracteatum, Macroptilium erythroloma, Macroptilium gracile, Macroptilium lathyroides, and Macroptilium martii) from the two main clades that form the genus. Heterochromatin distribution was investigated with chromomycin A3 (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization was used to localize the 5S and 35S ribosomal DNA (rDNA) sites. Single copy bacterial artificial chromosomes (BACs) previously mapped in the related genera Phaseolus L. and Vigna Savi were used to establish chromosome orthologies and to investigate possible rearrangements among species. CMA+/DAPI− bands were observed, mostly associated with rDNA sites. Additional weak, pericentromeric bands were observed on several chromosomes. Although karyotypes were similar, species could be differentiated mainly by the number and position of the 5S and 35S rDNA sites. BAC markers demonstrated conserved synteny of the main rDNA sites on orthologous chromosomes 6 and 10, as previously observed for Phaseolus and Vigna. The karyotypes of the six species could be differentiated, shedding light on its karyotype evolution. [ABSTRACT FROM AUTHOR]

Background: Cones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the "CreERT2" sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreERT2 mice with different cone cell specificities. Results: These models (Gnat2CreERT2, Arr3T2ACreERT2, and Arr3P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2CreERT2, 40% in Arr3T2ACreERT2, and 100% in Arr3P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript. Conclusions: The Arr3P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models. [ABSTRACT FROM AUTHOR]

Further studies will be necessary to elucidate the effects of TREM2 variants and targeting TREM2 function within the context of A -induced tau seeding and spreading as well as in the phase of tauopathy that is closely linked with neurodegeneration. What is it about the H157Y mutation and relationship between microglia, TREM2, sTREM2 and A that results in decreased A when assessed at a late stage of disease? Most notably, rare variants, such as the R47H, R62H and H157Y mutations, in the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) gene increase risk for late-onset AD [[1]]. Keywords: Alzheimer's disease; TREM2; Amyloid-beta; Microglia EN Alzheimer's disease TREM2 Amyloid-beta Microglia 1 4 4 04/06/23 20230405 NES 230405 Recent genetic studies have highlighted the central role of the innate immune system in Alzheimer's disease (AD) by identifying several risk variants in genes that are exclusively expressed within microglia in the brain. [Extracted from the article]

Background: Reverse genetics systems to rescue viruses from modified DNA are useful tools to investigate the molecular mechanisms of viruses. The COVID‐19 pandemic prompted the development of several reverse genetics systems for SARS‐CoV‐2. The circular polymerase extension reaction (CPER) method enables the rapid generation of recombinant SARS‐CoV‐2; however, such PCR‐based approaches could introduce unwanted mutations due to PCR errors. Methods: To compare the accuracy of CPER and a classic reverse genetics method using bacterial artificial chromosome (BAC), SARS‐CoV‐2 Wuhan/Hu‐1/2019 was generated five times using BAC and five times using CPER. These 10 independent virus stocks were then deep sequencing, and the number of substitutions for which the frequency was greater than 10% was counted. Results: No nucleotide substitutions with a frequency of greater than 10% were observed in all five independent virus stocks generated by the BAC method. In contrast, three to five unwanted nucleotide substitutions with a frequency of more than 10% were detected in four of the five virus stocks generated by the CPER. Furthermore, four substitutions with frequencies greater than 20% were generated in three virus stocks by using the CPER. Conclusions: We found that the accuracy of the CPER method is lower than that of the BAC method. Our findings suggest care should be used when employing the CPER method. [ABSTRACT FROM AUTHOR]

For the past three decades, the porcine epidemic diarrhea virus (PEDV) has remained an enormous threat to the South Korean swine industry. The scarcity of an effective method for manipulating viral genomes has impeded research progress in PEDV biology and vaccinology. Here, we report the development of reverse genetics systems using two novel infectious full-length cDNA clones of a Korean highly pathogenic-G2b strain, KNU-141112, and its live attenuated vaccine strain, S DEL5/ORF3, in a bacterial artificial chromosome (BAC) under the control of a eukaryotic promoter. Direct transfection of cells with each recombinant BAC clone induced cytopathic effects and produced infectious progeny. The reconstituted viruses, icKNU-141112 and icS DEL5/ORF3, harboring genetic markers, displayed phenotypic and genotypic properties identical to their respective parental viruses. Using the DNA-launched KNU-141112 infectious cDNA clone as a backbone, two types of recombinant viruses were generated. First, we edited the open reading frame 3 (ORF3) gene, as cell-adapted strains lose full-length ORF3, and replaced this region with an enhanced green fluorescent protein (EGFP) gene to generate icPEDV-EGFP. This mutant virus presented parental virus-like growth kinetics and stably retained robust EGFP expression, indicating that ORF3 is dispensable for PEDV replication in cell culture and is a tolerant location for exogeneous gene acceptance. However, the plaque size and syncytia phenotypes of ORF3-null icPEDV-EGFP were larger than those of icKNU-141112 but similar to ORF3-null icS DEL5/ORF3, suggesting a potential role of ORF3 in PEDV cytopathology. Second, we substituted the spike (S) gene with a heterologous S protein, designated S51, from a variant of interest (VOI), which was the most genetically and phylogenetically distant from KNU-141112. The infectious recombinant VOI, named icPEDV-S51, could be recovered, and the rescued virus showed indistinguishable growth characteristics compared to icKNU-141112. Virus cross-neutralization and structural analyses revealed antigenic differences in S between icKNU-141112 and icPEDV-S51, suggesting that genetic and conformational changes mapped within the neutralizing epitopes of S51 could impair the neutralization capacity and cause considerable immune evasion. Collectively, while the established molecular clones afford convenient, versatile platforms for PEDV genome manipulation, allowing for corroborating the molecular basis of viral replication and pathogenesis, they also provide key infrastructural frameworks for developing new vaccines and coronaviral vectors. [ABSTRACT FROM AUTHOR]

Background: HAP1, a near-haploid human leukemic cancer cell line is often used in combination with CRISPR-Cas9 gene editing technology for genetic screens. HAP1 carries the Philadelphia chromosome (Ph) and an additional ~ 30 Mb fragment of chromosome 15 inserted into chromosome 19. The potential use of an in vitro cell line as a model system in biomedical research studies depends on its ability to maintain genome stability. Being a cancer cell line with a near-haploid genome, HAP1 is prone to genetic instability, which is further compounded by its tendency to diploidise in culture spontaneously. Moreover, CRISPR-Cas9 gene editing coupled with prolonged in-vitro cell culturing has the potential to induce unintended 'off-target' cytogenetic mutations. To gain an insight into chromosomal instability (CIN) and karyotype heterogeneity, 19 HAP1 cell lines were cytogenetically characterised, 17 of which were near-haploids and two double-haploids, using multiplex fluorescence in situ hybridisation (M-FISH), at single cell resolution. We focused on novel numerical (N) and structural (S) CIN and discussed the potential causal factors for the observed instability. For each cell line we examined its ploidy, gene editing status and its length of in-vitro cell culturing. Results: Sixteen of the 19 cell lines had been gene edited with passage numbers ranging from 10 to 35. Diploidisation in 17 near-haploid cell lines ranged from 4 to 35% and percentage of N- and S-CIN in [1n] and [2n] metaphases ranged from 7 to 50% with two cell lines showing no CIN. Percentage of cells with CIN in the two double-haploid cell lines were 96% and 100% respectively. The most common S-CIN observed was deletion followed by translocation of both types, non-reciprocal and Robertsonian. Interestingly, we observed a prevalence of S-CIN associated with chromosome 13 in both near-and double-haploid cell lines, with a high incidence of Robertsonian translocation involving chromosome 13. Furthermore, locus-specific BAC (bacterial artificial chromosome) FISH enabled us to show for the first time that the additional chromosome 15 fragment is inserted into the p-arm rather than the q-arm of chromosome 19 of the HAP1 genome. Conclusion: Our study revealed a high incidence of CIN leading to karyotype heterogeneity in majority of the HAP1 cell lines with the number of chromosomal aberrations varying between cell lines. A noteworthy observation was the high frequency of structural chromosomal aberrations associated with chromosome 13. We showed that CRISPR-Cas9 gene editing technology in combination with spontaneous diploidisation and prolonged in-vitro cell culturing is potentially instrumental in inducing further chromosomal rearrangements in the HAP1 cell lines with existing CIN. We highlight the importance of maintaining cell lines at low passage and the need for regular monitoring to prevent implications in downstream applications. Our study also established that the additional fragment of chromosome 15 in the HAP1 genome is inserted into chromosome 19p rather than 19q. [ABSTRACT FROM AUTHOR]

Herpesvirus saimiri (HVS) is discussed as a possible vector in gene therapy. In order to create a self-repairing HVS vector, the F plasmid vector moiety of the bacterial artificial chromosome (BAC) was transposed via Red recombination into the virus genes ORF22 or ORF29b, both important for virus replication. Repetitive sequences were additionally inserted, allowing the removal of the F-derived sequences from the viral DNA genome upon reconstitution in permissive epithelial cells. Moreover, these self-repair-enabled BACs were used to generate deletion variants of the transforming strain C488 in order to minimalize the virus genome. Using the en passant mutagenesis with two subsequent homologous recombination steps, the BAC was seamlessly manipulated. To ensure the replication capacity in permissive monkey cells, replication kinetics for all generated virus variants were documented. HVS variants with increased insert capacity reached the self-repair within two to three passages in permissive epithelial cells. The seamless deletion of ORFs 3/21, 12–14, 16 or 71 did not abolish replication competence. Apoptosis induction did not seem to be altered in human T cells transformed with deletion variants lacking ORF16 or ORF71. These virus variants form an important step towards creating a potential minimal virus vector for gene therapy, for example, in human T cells. [ABSTRACT FROM AUTHOR]

Conventional vector systems, including plasmid-based vectors, are mainly used in mammalian cells for the production of biopharmaceuticals. Plasmid-based vectors express transgene by the random integration of recombinant transgenes into the genome. Transgene expression is greatly influenced by the surrounding chromatin, and in most cases, expression is weak and tends to be suppressed over time. Therefore, a novel strategy is required to create clones that maintain increased transgene expression. In this study, we used a bacterial artificial chromosome (BAC) containing the Rosa26 locus, which allows constitutive and ubiquitous gene expression. Moreover, we improved the Rosa26 BAC-mediated expression system by incorporating the Tol2 transposon system with helper vector, resulting in improved efficiency of protein production and maintained productivity even in single-cell clones. Furthermore, the recombinant Rosa26 BAC was improved in terms of protein productivity by using helper mRNA instead of helper vector. Finally, establishment of an optimal molar ratio between the recombinant Rosa26 BAC and helper mRNA helped to achieve maximal protein productivity. Taken together, our results provide an effective strategy for improving BAC-based expression systems for biopharmaceutical production. [ABSTRACT FROM AUTHOR]

Summary: Wheat stem (or black) rust, caused by Puccinia graminis f. sp. tritici (Pgt), has been historically among the most devastating global fungal diseases of wheat. The recent occurrence and spread of new virulent races such as Ug99 have prompted global efforts to identify and isolate more effective stem rust resistance (Sr) genes. Here, we report the map‐based cloning of the Ug99‐effective SrTm5 gene from diploid wheat Triticum monococcum accession PI 306540 that encodes a typical coiled‐coil nucleotide‐binding leucine‐rich repeat protein. This gene, designated as Sr22b, is a new allele of Sr22 with a rare insertion of a large (13.8‐kb) retrotransposon into its second intron. Biolistic transformation of an ~112‐kb circular bacterial artificial chromosome plasmid carrying Sr22b into the susceptible wheat variety Fielder was sufficient to confer resistance to stem rust. In a survey of 168 wheat genotypes, Sr22b was present only in cultivated T. monococcum subsp. monococcum accessions but absent in all tested tetraploid and hexaploid wheat lines. We developed a diagnostic molecular marker for Sr22b and successfully introgressed a T. monococcum chromosome segment containing this gene into hexaploid wheat to accelerate its deployment and pyramiding with other Sr genes in wheat breeding programmes. Sr22b can be a valuable component of gene pyramids or transgenic cassettes combining different resistance genes to control this devastating disease. [ABSTRACT FROM AUTHOR]

The green peach aphid (GPA), Myzus persicae , is a polyphagous, sap-sucking aphid and a vector of many plant viruses. In peach, Prunus persica , three individual dominant GPA resistance loci have been genetically defined (Rm1 – 3), but knowledge of the underlying genes is limited. In this study, we focused on the Rm3 locus. Bulk segregant analysis (BSA) mapping in segregating progeny populations delimited Rm3 to an interval spanning 160 kb containing 21 genes on chromosome 1. RNA-seq data provided no evidence of candidate genes, but chromosomal structural variations were predicted around a nucleotide-binding site–leucine-rich repeat (NLR) gene (ppa000596m) within the Rm3 fine-mapping interval. Following bacterial artificial chromosome (BAC) library construction for a GPA-resistant peach cultivar and the sequencing of three target BAC clones, a chromosomal structural variation encompassing two novel TIR–NLR-class disease resistance (R) protein-coding genes was identified, and the expressed NLR gene (NLR1) was identified as a candidate for M. persicae resistance. Consistent with its proposed role in controlling GPA resistance, NLR1 was only expressed in the leaves of resistant peach phenotypes. A molecular marker that was designed based on the NLR1 sequence co-segregated with the GPA-resistant phenotype in four segregating populations, 162 peach cultivars, and 14 wild relatives, demonstrating the dominant inheritance of the Rm3 locus. Our findings can be exploited to facilitate future breeding for GPA-resistance in peach. [ABSTRACT FROM AUTHOR]

Aging is often associated with cognitive decline and recurrent cellular and molecular impairments. While life-long caloric restriction (CR) may delay age-related cognitive deterioration as well as the onset of neurologic disease, recent studies suggest that late-onset, short-term intermittent fasting (IF), may show comparable beneficial effects as those of life-long CR to improve brain health. We used a new optogenetic aging model to study the effects of late-onset (.18 months), short-term (four to six weeks) IF on age-related changes in GABAergic synaptic transmission, intracellular calcium (Ca2+) buffering, and cognitive status. We used male mice from a bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R [VGAT-ChR2(H134R)-EYFP] in a reduced synaptic preparation that allows for specific optogenetic light stimulation on GABAergic synaptic terminals across aging. We performed quantal analysis using the method of failures in this model and show that short-term IF reverses the age-related decrease in quantal content of GABAergic synapses. Likewise, short-term IF also reversed age-related changes in Ca2+ buffering and spontaneous GABAergic synaptic transmission in basal forebrain (BF) neurons of aged mice. Our findings suggest that late-onset shortterm IF can reverse age-related physiological impairments in mouse BF neurons but that four weeks IF is not sufficient to reverse age-related cognitive decline. [ABSTRACT FROM AUTHOR]

We introduce SilkBase as an integrated database for transcriptomic and genomic resources of the domesticated silkworm Bombyx mori and related species. SilkBase is the oldest B. mori database that was originally established as the expressed sequence tag database since 1999. Here, we upgraded the database by including the datasets of the newly assembled B. mori complete genome sequence, predicted gene models, bacterial artificial chromosome (BAC)-end and fosmid-end sequences, complementary DNA (cDNA) reads from 69 libraries, RNA-seq data from 10 libraries, PIWI-interacting RNAs (piRNAs) from 13 libraries, ChIP-seq data of 9 histone modifications and HP1 proteins and transcriptome and/or genome data of four B. mori -related species, i.e. Bombyx mandarina, Trilocha varians, Ernolatia moorei and Samia ricini. Our new integrated genome browser easily provides a snapshot of tissue- and stage-specific gene expression, alternative splicing, production of piRNAs and histone modifications at the gene locus of interest. Moreover, SilkBase is useful for performing comparative studies among five closely related lepidopteran insects. Database URL : https://silkbase.ab.a.u-tokyo.ac.jp [ABSTRACT FROM AUTHOR]

Duck plague virus (DPV) can cause high morbidity and mortality in many waterfowl species within the order Anseriformes. The DPV genome contains 78 open reading frames (ORFs), among which the LORF2, LORF3, LORF4, LORF5, and SORF3 genes are unique genes of avian herpesvirus. In this study, to investigate the role of this unique LORF5 gene in DPV proliferation, we generated a recombinant virus that lacks the LORF5 gene by a two-step red recombination system, which cloned the DPV Chinese virulent strain (DPV CHv) genome into a bacterial artificial chromosome (DPV CHv-BAC); the proliferation law of LORF5-deleted mutant virus on DEF cells and the effect of LORF5 gene on the life cycle stages of DPV compared with the parent strain were tested. Our data revealed that the LORF5 gene contributes to the cell-to-cell transmission of DPV but is not relevant to virus invasion, replication, assembly, and release formation. Taken together, this study sheds light on the role of the avian herpesvirus-specific gene LORF5 in the DPV proliferation life cycle. These findings lay the foundation for in-depth functional studies of the LORF5 gene in DPV or other avian herpesviruses. [ABSTRACT FROM AUTHOR]

Recombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone. Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days. Overall, our new strategy may provide a new avenue for BAC recombineering, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration. [ABSTRACT FROM AUTHOR]

With the advent of chromatin‐interaction maps, chromosome‐level genome assemblies have become a reality for a wide range of organisms. Scaffolding quality is, however, difficult to judge. To explore this gap, we generated multiple chromosome‐scale genome assemblies of an emerging wild animal model for carcinogenesis, the California sea lion (Zalophus californianus). Short‐read assemblies were scaffolded with two independent chromatin interaction mapping data sets (Hi‐C and Chicago), and long‐read assemblies with three data types (Hi‐C, optical maps and 10X linked reads) following the "Vertebrate Genomes Project (VGP)" pipeline. In both approaches, 18 major scaffolds recovered the karyotype (2n = 36), with scaffold N50s of 138 and 147 Mb, respectively. Synteny relationships at the chromosome level with other pinniped genomes (2n = 32–36), ferret (2n = 34), red panda (2n = 36) and domestic dog (2n = 78) were consistent across approaches and recovered known fissions and fusions. Comparative chromosome painting and multicolour chromosome tiling with a panel of 264 genome‐integrated single‐locus canine bacterial artificial chromosome probes provided independent evaluation of genome organization. Broad‐scale discrepancies between the approaches were observed within chromosomes, most commonly in translocations centred around centromeres and telomeres, which were better resolved in the VGP assembly. Genomic and cytological approaches agreed on near‐perfect synteny of the X chromosome, and in combination allowed detailed investigation of autosomal rearrangements between dog and sea lion. This study presents high‐quality genomes of an emerging cancer model and highlights that even highly fragmented short‐read assemblies scaffolded with Hi‐C can yield reliable chromosome‐level scaffolds suitable for comparative genomic analyses. [ABSTRACT FROM AUTHOR]

The phylogenetic position and taxonomic status of Rhynchocyclidae (Aves: Passeriformes) have been the subject of debate since their first description. In most models, Rhynchocyclidae represents a subfamily-level taxon placed within the Tyrant Flycatchers (Tyrannidae). Considering that this classification does not include cytotaxonomic characters, we tested the hypothesis that the chromosome organization of Rhynchocyclidae members differs from that of Tyrannidae. Hence, we selected two species, Tolmomyias sulphurescens, and Pitangus sulphuratus, representing Rhynchocyclidae and Tyrannidae, respectively. Results revealed a diploid number (2n) of 60 in T. sulphurescens and 2n = 80 in P. sulphuratus, indicating significant chromosomal differences. Chromosome mapping of Gallus gallus (GGA) and Taeniopygia guttata bacterial artificial chromosome (BAC) corresponding to chromosomes GGA1-28 (except 16) revealed that the genome evolution of T. sulphurescens involved extensive chromosome fusions of macrochromosomes and microchromosomes. On the other hand, P. sulphuratus retained the ancestral pattern of organization of macrochromosomes (except the centric fission involving GGA1) and microchromosomes. In conclusion, comparing our results with previous studies in Tyrant Flycatchers and allies indicates that P. sulphuratus has similar karyotypes to other Tyrannidae members. However, T. sulphurescens does not resemble the Tyrannidae family, reinforcing family status to the clade named Rhynchocyclidae. [ABSTRACT FROM AUTHOR]