Identical DNA Sequences Research Articles

Distribution and environmental preferences of Saprolegnia and Leptolegnia (Oomycota) species in fish hatcheries in Hungary

We studied for the first time the abundance and species distribution of water molds (Oomycota, Saprolegniales) in fish hatcheries in Hungary, with a focus on Saprolegnia and Leptolegnia spp. A total of 182 water mold isolates were obtained from different developmental stages of fish (eggs, fry, and adults) and water samples collected during the spawning season of cyprinids and salmonids. Of the ten water mold species identified based on their ITS–rDNA sequences, S. parasitica, S. ferax, and S. australis were the most abundant. Statistical analyses of species proportions in relation to the sample source and host preference revealed that S. parasitica was significantly more abundant in the host than in water samples, whereas the opposite trend was observed for S. ferax and S. australis. In addition, our results confirmed previous findings that S. australis and S. ferax prefer warm water (+21–24 °C) and that S. parasitica has a wide temperature tolerance (+3–24 °C). The DNA sequence identity analysis showed that the species demarcation criterion for Saprolegnia spp. was approximately 98 % identity, based on ITS–rDNA sequences. Furthermore, our phylogenetic analyses suggest that taxonomic revision of Leptolegnia spp. is necessary.

Aquatic nitrous oxide reductase gene (nosZ) phylogeny and environmental distribution.

Nitrous oxide (N2O) is a potent greenhouse gas and a major cause of ozone depletion. One-third of atmospheric N2O originates in aquatic environments. Reduction of N2O to dinitrogen gas (N2) requires the nitrous oxide reductase enzyme, which is encoded by the gene nosZ. Organisms that contain nosZ are the only known biological sinks of N2O and are found in diverse genera and a wide range of environments. The two clades of nosZ (Clade I and II) contain great diversity, making it challenging to study the population structure and distribution of nosZ containing organisms in the environment. A database of over 11,000 nosZ sequences was compiled from NCBI (representing diverse aquatic environments) and unpublished sequences and metagenomes (primarily from oxygen minimum zones, OMZs, where N2O levels are often elevated). Sequences were clustered into archetypes based on DNA and amino acid sequence identity and their clade, phylogeny, and environmental source were determined. Further analysis of the source and environmental distribution of the sequences showed strong habitat separation between clades and phylogeny. Although there are more Clade I nosZ genes in the compilation, Clade II is more diverse phylogenetically and has a wider distribution across environmental sources. On the other hand, Clade I nosZ genes are predominately found within marine sediment and are primarily from the phylum Pseudonomonadota. The majority of the sequences analyzed from marine OMZs represented distinct phylotypes between different OMZs showing that the nosZ gene displays regional and environmental separation. This study expands the known diversity of nosZ genes and provides a clearer picture of how the clades and phylogeny of nosZ organisms are distributed across diverse environments.

Borrelia nietonii sp. nov.: Relapsing Fever Spirochetes Transmitted By the Tick Ornithodoros hermsi Designated Previously as Borrelia hermsii Genomic Group II.

Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined. Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined. Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86-95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3' end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined. Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.

Differences of circular RNA expression profiles between monozygotic twins' blood, with the forensic application in bloodstain and saliva

Monozygotic twins (MZTs) possess identical genomic DNA sequences and are usually indistinguishable through routine forensic DNA typing methods, which can be relevant in criminal and paternity cases. Recently, novel epigenetic methods involving DNA methylation and microRNA analysis have been introduced to differentiate MZTs. In this study, we explore the potential of using epigenetic markers, specifically circular RNAs (circRNAs), a type of non-coding RNA (ncRNA), to identify MZTs, and investigate the unique expression patterns of circRNAs within pairs of MZTs, enabling effective differentiation. Epigenetics regulates gene expression at the post-transcriptional level and plays a crucial role in cell growth and aging. CircRNAs, a recently characterized subclass of ncRNA, have a distinct covalent loop structure without the typical 5′ cap or 3′ tail. They have been reported to modulate various cellular processes and play roles in embryogenesis and eukaryotic development. To achieve this, we conducted a comprehensive circRNA sequencing analysis (circRNA-seq) using total RNA extracted from the blood samples of five pairs of MZTs. We identified a total of 15,257 circRNAs in all MZTs using circRNA-seq. Among them, 3, 21, 338, and 2967 differentially expressed circRNAs (DEcircRNAs) were shared among five, four, three, and two pairs of MZTs, respectively. Subsequently, we validated twelve selected DEcircRNAs using real-time quantitative polymerase chain reaction (RT-qPCR) assays, which included hsa_circ_0004724, hsa_circ_0054196, hsa_circ_004964, hsa_circ_0000591, hsa_circ_0005077, hsa_circ_0054853, hsa_circ_0054716, hsa_circ_0002302, hsa_circ_0004482, hsa_circ_0001103, novel_circ_0030288 and novel_circ_0056831. Among them, hsa_circ_0005077 and hsa_circ_0004482 exhibited the best performance, showing differences in 7 out of 10 pairs of MZTs. These twelve differentially expressed circRNAs also demonstrated strong discriminative power when tested on saliva samples from 10 pairs of MZTs. Notably, hsa_circ_0004724 displayed differential expression in 8 out of 10 pairs of MZTs in their saliva. Additionally, we evaluated the detection sensitivity, longitudinal temporal stability, and suitability for aged bloodstains of these twelve DEcircRNAs in forensic scenarios. Our findings highlight the potential of circRNAs as molecular markers for distinguishing MZTs, emphasizing their suitability for forensic application.

Occurrence and seasonal variation of Perkinsus sp. Infection in wild mollusk populations from coastal waters of Qingdao, northern China

Perkinsosis has been recognized as one of the major threats to natural and farmed bivalve populations, many of which are of commercial as well as environmental significance. Three Perkinsus species have been identified in China, and the Manila clam (Ruditapes philippinarum) was the most frequently infected species in northern China. Although the occurrence and seasonal variation of Perkinsus spp. have previously been examined, the pathological characteristics of these infections in wild Manila clams and sympatric species in China have seldom been reported. In the present study, the prevalence and intensity of Perkinsus infection in wild populations of Manila clams and 10 sympatric species from three sites were investigated by Ray’s fluid thioglycolate medium (RFTM) assay seasonally across a single year. Perkinsus infection was only identified in Manila clams, with a high prevalence (274/284 = 96.48 %) and low intensity (89.8 % with a Mackin value ≤ 2, suggesting generally low-intensity infections) throughout the year. Heavily infected clams were mainly identified in Tianheng in January, which displayed no macroscopic signs of disease. An overview of the whole visceral mass section showed that the trophozoites mostly aggregated in gills and connective tissue of the digestive tract, to a lesser extent in the mantle and foot, and even less frequently in adductor muscle and connective tissues of the gonad. PCR and ITS-5.8S rRNA sequencing of 93 representative RFTM-positive samples revealed a 99.69 to 100 % DNA sequence identity to Perkinsus olseni. Unexpectedly, significantly higher infection intensities were usually identified in January and April when the Condition Index (CI) was relatively high. We propose that factors associated with the anthropogenic harvesting pressure and irregular disturbances should be responsible for the uncommon seasonal infection dynamics of perkinsosis observed in the present study.

Wild Chaga (Inonotus obliquus) modulates inflammation, neural cell survival and inhibits proliferation of cancer cells

Background and AimInonotus obliquus (Ach. Ex. Pers), commonly known as Chaga, is a parasitic fungus that grows on birch trees. Indigenous North Americans and Northern Eurasians have used Chaga tea as a folk medicine for centuries, and there are currently significant interests in commercializing Chaga products, obtained either from conks (sclerotia) collected from nature or from laboratory cultured mycelia. The cultured mycelia's identity to date is based solely on chemical profiling and the morphologic characteristics; and genome identity are often overlooked and unreported in the literature. The aims of this study include: 1. To authenticate mycelium isolates derived from a Chaga sclerotium; 2. To determine Chaga sclerotium's bioactivities unreported previously. Experimental proceduresIdentification of Chaga conk was carried out by chemical analyses, culture characteristics, and genome identity. The Chaga extracts’ bioactivities were evaluated via effects on cytokine and reactive oxygen species production on normal and cancer cell survival and proliferation. Results and ConclusionThe features of this mycelium isolate conformed to I. obliquus mycology, with > 99 % DNA sequence identity to the known GenBank Chaga Ribosomal Internal Spacer (ITS) sequence. Water extracts of Chaga were effective in protecting against oxidative stress in mouse neuroblastoma-spinal motor neuron NSC-34 cells and inhibited the proliferation of human brain glioblastoma DBTRG-05MG and pancreatic ductal adenocarcinoma PANC-1 cancer cell lines in vitro. In addition, the water extract potently stimulated inflammatory cytokines release from human blood cells, consistent with the biological response modifier features of mushroom beta- glucan polysaccharides.

Experimental exposure of rainbow trout eggs to water mould, Saprolegnia parasitica (Oomycota): intraspecific differences in pathogenicity

AbstractIn in vivo infection trials, rainbow trout eyed eggs were exposed to three Saprolegnia parasitica (Oomycota) isolates, which differed in biological and genetic characteristics. Infection prevalence, hatching rate, hatching dynamics of fish eggs were quantified, and the study was complemented with histopathology and phylogenetic analyses. We experimentally detected intraspecific differences in the pathogenicity of S. parasitica on rainbow trout eggs. The isolate from rainbow trout eggs was the most virulent to eggs of the same host, whereas isolates from carp skin and fry did not cause as much damage to the eggs. Comparing the outcome of two experimental settings, we confirmed that invasion of fish eggs is more effective by hyphae growth than by the actively moving zoospores. In addition, our findings highlighted that S. parasitica isolates with 100% identical ITS DNA sequences, could differ significantly in virulence. These isolates can be clearly distinguished based on a 650-bp DNA fragment of the DNA-directed RNA polymerase II subunit (RPB2) gene.

Fruit rot causing by Athelia rolfsii (Sclerotium rolfsii) on jackfruit (Artocarpus heterophyllus) in China.

Artocarpus heterophyllus, known as jackfruit, was a tropical fruit and cultivated extensively as nutritional and medicinal properties in southern China in recent year. During July 2022, fruit rot was observed on the fruits at the bottom of jackfruit trees in an orchard in Zhanjiang, Guangdong (N21°9' 27" E110°17' 54") 3-4 days after typhoon. The incidence rate of fruit was about 0.3%. The initial symptom was white mycelia appearing on the surface of fruits. Mycelia with rhizomorphs spread rapidly over the fruits, formed white, often fan-shaped mats with the rapeseed size sclerotia. The infected fruits were water-soaked, quickly became rotten, and fell off. Sclerotia from disease fruits were incubated on PDA with 50 mg/L ampicillin at 25-28℃ in the dark for 2 days. Hyphae tips were transferred to get the purified isolates. Colonies with a radial growth rate of 23.2 mm/day had abundant aerial mycelia and profuse sclerotia on PDA. Hyphae of the isolates were transparent, branched, with clamp connections at septa, usually 2.9-8.3 µm (Ave. 5.8 µm) (n>30) wide. Aerial mycelia were whitish-cottony, with many narrow rhizomorphs. Spherical sclerotia developed at about 10 days after incubation, and gradually changed from white to tan-to-dark brown, and mature sclerotia were about 1.7 mm in size. The morphological characteristics was similar to those of Sclerotium rolfsii (teleomorph: Athelia rolfsii). To accurately identify the fungus, the internal transcribed spacer gene (ITS) and large subunit rRNA gene (LSU) of isolate CASS-BLM-1 were PCR amplified with primer pairs ITS1/ITS4 (White et al 1990) and V9G/LR5 (Klaubauf et al 2014). The amplicons were sequenced and deposited in GenBank with accession number OP535473 (ITS) and OP535474 (LSU). BLASTn results showed that the nucleotide sequences of ITS and LSU had high identity with corresponding sequences of A. rolfsii isolates CBS 191.62 (ITS: MH858139, 472/474(99.58%); LSU: MH869724, 882/885(99.66%)) (Vu et al 2019). Phylogenetic analysis based on ITS sequence data was obtained according to maximum likelihood method using MEGA analysis software, CASS-BLM1 was grouped in A. rolfsii clade with 100% bootstrap support value. Based on morphology and DNA sequences, the fungus was identified as A. rolfsii (anamorph: S. rolfsii). To fulfil Koch's postulates, healthy fruits on the tree and detached fruits were inoculated with 7-day-old sclerotia of isolate CASS-BLM1. Five unwound sites and five wound sites with a sterile needle were tested on each fruit and a sclerotium was put at each site. Fruits not inoculated with sclerotia were used as control the test was repeated three times. All fruit were enclosed in transparent plastic bags with sterile absorbent cotton moistened with sterile distilled water. The indoor and outdoor temperatures ranged from 25 to 30 ℃. Three days later, white mycelia were observed on all inoculation sites, and 5 days later, the inoculated fruits began to rot, while control fruits remained healthy. The same fungus with identical morphology and DNA sequences was re-isolated from the inoculated sites. Previously, A. rolfsii was reported to cause fruit rot disease on jackfruit in Bangladesh (Elahi et al 2021), this is the first report of A. rolfsii causing fruit rot on jackfruit in China. A. rolfsii is suitable for high temperature and humidity environment (Punja 1985), this report will help farmers to diagnose this disease, especially to strengthen the disease prevention during the typhoon season.

Detection of Bartonella DNA in Yellow Flies, Lone Star Ticks, and a Human Patient with Concurrent Evidence of Borrelia burgdorferi Infection in Northeast Florida, USA.

Background: Bartonella species and Borrelia burgdorferi sensu lato (Bbsl) are emerging zoonotic pathogens. The vectors and frequency of infections with both pathogen groups in the southern United States is understudied. This study describes an investigation of Bartonella and Bbsl in yellow flies collected at a residence in northeast Florida, USA, that led to subsequent discoveries of both organisms in lone star ticks (Amblyomma americanum) and a human patient. Materials and Methods: DNA samples from flies, ticks, and human patient blood specimens were tested via polymerase chain reaction assays for Bartonella or Bbsl species. DNA sequences were compared to reference strains for identification and characterization. Results: An exploratory investigation of arthropod-borne pathogens in yellow flies collected at a residence in northeast Florida revealed the presence of uncharacterized Bartonella species DNA sequences similar to ones previously detected in two lone star ticks from Virginia. Subsequent testing of several lone star ticks from the area detected similar sequences of Bartonella in three ticks. Testing of stored blood samples from a resident of the site, who had experienced chronic relapsing and remitting symptoms for over a decade, identified nearly identical Bartonella DNA sequences in multiple samples collected over a 10-year period. Two lone star ticks and several samples from the same patient and time period also tested positive for Bo. burgdorferi DNA, suggesting possible long-term coinfection of the patient with both organisms. Conclusion: This investigation identified highly similar Bartonella DNA sequences in yellow flies, lone star ticks, and a human patient in northeast Florida. Similarly, Bo. burgdorferi DNA was detected in two lone star ticks and multiple specimens from the patient. Positive PCR results from archived patient blood samples documented the presence of both organisms at multiple time points over more than a decade. More studies on human patients with chronic undefined illness and on the presence of Bartonella and Bbsl in hematophagous arthropods and animal hosts in the southeastern United States are needed.

Dianthus × belkinii (Caryophyllaceae), a new hybrid taxon from Altai

Dianthus × belkinii is an intersectional hybrid newly described from the Republic of Altai (Talduair massif). This taxon was also noted in Mongolia (Bayan-Ulegei aimag). The article describes the morphological features of the new taxon and discusses its relationship with parental species. The described hybrid has intermediate morphological features between the parental species, most notable are the fringed corollas. The stems are sparsely branched, relatively tall and similar to those of D. superbus. The calyx lobes and bracts are transitional between D. superbus and D. versicolor. The limb of the corolla is cut for ⅓–¼ of its length into a few lobes. Cytometry data showed an identical ploidy level of the new taxon and its putative parent species. The average value of the relative DNA content is almost intermediate, however, somewhat closer to D. versicolor. The ITS sequences of D. × belkinii show characteristic double peaks, which may point to a hybridogeneous origin of the plants. Identical chloroplast DNA sequences with D. versicolor but differences to D. superbus suggests that D. versicolor may be the maternal parent of this hybrid. Based on these polymorphisms, it is possible to state that the ITS sequence and D. versicolor × superbus itself are of a hybrid nature, where D. versicolor and D. superbus are the parent forms.

Comparison of Genotypic and Phenotypic Predictions for Heavy Metal Resistance in Salmonella enterica and Escherichia coli

Salmonella enterica and Escherichia coli are two pathogenic bacteria of worldwide importance that can infect the gastrointestinal tract. Contamination in the food supply chain is an area of concern. Animal feed may be supplemented with essential trace elements, which function as nutritional additives to promote growth & health and optimize production. Bacteria have acquired many metal resistance genes to adapt to the exposure of metals. In this study, our objectives were to evaluate in S. enterica and E. coli, the correlation between the resistance genotype and phenotype to certain heavy metals, and the ability of conjugative plasmids to transfer antimicrobial resistance genes (AMRGs) and heavy metal resistance genes (HMRGs). A total of 10 strains, five S. enterica and five E. coli, were used for this study. Minimal inhibitory concentrations (MICs) were determined for heavy metals: copper, silver, arsenic, and tellurite. The tested isolates showed resistance to copper (5/10; 50%), arsenic (7/10; 70%), and silver (9/10; 90%). Cohen’s Kappa statistics were used to analyze genotype to phenotype agreements. Among the 10 strains sampled, the accordance between geno- and phenotypic heavy metal resistance was fair for copper (kappa = 0.4), none to slight for arsenic (kappa = 0.19) and tellurite (kappa = 0), and no agreement for silver (kappa = -0.19). The transfer of HMRGs was determined in a conjugation experiment performed for all five Salmonella strains as donors using mixed broth cultures. Transconjugants were obtained only from the genotypically tellurite-resistant strain PSU-3260, which yielded a transfer frequency of 10⁻³ transconjugants per donor. In such strain, the tellurite-resistant genes reside on an IncHI2-type plasmid that shares high DNA sequence identity with known HMRG-disseminating Salmonella plasmids. Our results indicated no considerable correlation between the geno- and phenotypic resistance towards heavy metals in the sampled S. enterica and E. coli. The necessity of research in this area is supported by the lack of standardized protocols and MIC clinical breakpoints for heavy metals. KEYWORDS: Heavy metal; resistance; Salmonella; E. coli; agriculture; genotype; phenotype; MIC

Molecular identification of the broodsacs from Leucochloridium passeri (Digenea: Leucochloridiidae) with a review of Leucochloridium species records in Taiwan.

Leucochloridium spp. have been established in Taiwan since the 1930s, with five species listed: Leucochloridium turdi, Leucochloridium passeri, Leucochloridium muscularae, Leucochloridium sime, and Leucochloridium taiwanese listed until now. Because of the similar morphology among the adults of the Leucochloridiidae family, the taxonomic status of most of them should be reconsidered. Broodsacs are the most distinct characteristic shared by Leucochloridium flukes, and L. passeri is, by far, the only one whose broodsacs have been described. In this study, broodsacs collected from an infected Succinea sp. (amber snails) in Taiwan were morphologically compared to previous descriptions and sequenced using both nuclear and mitochondrial genetic markers for identification. Our samples resembled broodsacs of L. passeri previously described in northern Taiwan and those recently collected in Okinawa, Japan. The conspecific status of the broodsacs collected in Taiwan and Okinawa was supported by the identical DNA sequences identified in this study. Broodsacs have rarely been reported in tropical and subtropical regions, although some Leucochloridium spp. have been recorded. In such cases, genetic markers will be crucial to link Leucochloridium broodsacs in snails (intermediate host) with their corresponding adult stage in birds (definitive host).

DNA sequence and chromatin modifiers cooperate to confer epigenetic bistability at imprinting control regions

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.

Molecular characterization of methicillin resistant Staphylococcus aureus isolated from hospitals environments and patients in Northern Palestine


 BACKGROUND: Staphylococcus aureus (S. aureus) is considered one of the most common pathogen to humans. Infections caused by this mocroorganism can be acquired through both hospital and community settings. This study was carried out to investigate molecular characterization of MRSA strains isolated from the patients and their environment in two hospitals (Rafidia hospital and Thabet hospital) inNorthern Palestine, and to determine the clonal identity between these strains and their possible contribution to nosocomial infections.
 METHODS: Two hundred sixty five swabbed samples were collected from these hospitals, S. aureus was isolated, antibiotic resistant genes were Panton–Valentin leukocidin (PVL) gene were detected and SCCmec and spA were typed by PCR and/or sequencing.
 RESULTS: The prevalence of MRSA among S. aureus isolates was 29% and 8.2% in Rafidia hospital and Thabet hospital, respectively. All strains resistant to oxacilllin disk were carried mecA gene. Majority of strains (84.6%) carried SCCmec type II (n = 11), type IVa and non-typeable were also detected. In addition, PVL was detected in 2 (14.3%) clinical strains. ERIC PCR patterns revealed that 2 strains recovered from patient bed and nasal swab isolated from Thabet Hospital were nontypeable, spA typing showed that they belonged to type t386 and have identical DNA sequences. Other 2 clinical isolates were spa typed, one belonged to clone t044, while the other is new clone not exist in database.
 CONCLUSIONS: Results may give evidence that environmental contamination possibly contributing to nosocomial infections.

Sister chromatid-sensitive Hi-C to map the conformation of replicated genomes.

Chromosome conformation capture (Hi-C) techniques map the 3D organization of entire genomes. How sister chromatids fold in replicated chromosomes, however, cannot be determined with conventional Hi-C because of the identical DNA sequences of sister chromatids. Here, we present a protocol for sister chromatid-sensitive Hi-C (scsHi-C) that enables the distinction of DNA contacts within individual sister chromatids (cis sister contacts) from those between sister chromatids (trans sister contacts), thereby allowing investigation of the organization of replicated genomes. scsHi-C is based on live-cell labeling of nascent DNA by the synthetic nucleoside 4-thio-thymidine (4sT), which incorporates into a distinct DNA strand on each sister chromatid because of semi-conservative DNA replication. After purification of genomic DNA and in situ Hi-C library preparation, 4sT is chemically converted into 5-methyl-cytosine in the presence of OsO4/NH4Cl to introduce T-to-C signature point mutations on 4sT-labeled DNA. The Hi-C library is then sequenced, and ligated fragments are assigned to sister chromatids on the basis of strand orientation and the presence of signature mutations. The ensemble of scsHi-C contacts thereby represents genome-wide contact probabilities within and across sister chromatids. scsHi-C can be completed in 2 weeks, has been successfully applied in HeLa cells and can potentially be established for any cell type that allows proper cell cycle synchronization and incorporation of sufficient amounts of 4sT. The genome-wide maps of replicated chromosomes detected by scsHi-C enable investigation of the molecular mechanisms shaping sister chromatid topologies and the relevance of sister chromatid conformation in crucial processes like DNA repair, mitotic chromosome formation and potentially other biological processes.

RFPlasmid: predicting plasmid sequences from short-read assembly data using machine learning.

Antimicrobial-resistance (AMR) genes in bacteria are often carried on plasmids and these plasmids can transfer AMR genes between bacteria. For molecular epidemiology purposes and risk assessment, it is important to know whether the genes are located on highly transferable plasmids or in the more stable chromosomes. However, draft whole-genome sequences are fragmented, making it difficult to discriminate plasmid and chromosomal contigs. Current methods that predict plasmid sequences from draft genome sequences rely on single features, like k-mer composition, circularity of the DNA molecule, copy number or sequence identity to plasmid replication genes, all of which have their drawbacks, especially when faced with large single-copy plasmids, which often carry resistance genes. With our newly developed prediction tool RFPlasmid, we use a combination of multiple features, including k-mer composition and databases with plasmid and chromosomal marker proteins, to predict whether the likely source of a contig is plasmid or chromosomal. The tool RFPlasmid supports models for 17 different bacterial taxa, including Campylobacter , Escherichia coli and Salmonella , and has a taxon agnostic model for metagenomic assemblies or unsupported organisms. RFPlasmid is available both as a standalone tool and via a web interface.

Simultaneous Production of Multiple Antimicrobial Compounds by Bacillus velezensis ML122-2 Isolated From Assam Tea Leaf [Camellia sinensis var. assamica (J.W.Mast.) Kitam.

Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum. The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75–100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin (mrsA), amylocyclicin (acnA), plipastatin (ppsA), and surfactin (srfAA) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea.

Development of a Bicistronic Anti-CD19/CD20 CAR Construct Including Optimization to Abrogate Retroviral Recombination Events

Anti-CD19 chimeric antigen receptor (CAR) T cells can eliminate lymphoma, but CD19 loss from lymphoma can cause anti-CD19 CAR T-cell treatment failure. To address CD19 loss, we designed a bicistronic construct encoding 2 CARs. The first CAR in the bicistronic construct was the anti-CD19 CAR Hu19-CD828Z, which incorporated a human anti-CD19 single-chain variable fragment (scFv) along with CD28 and CD3ζ domains (Brudno et al. Nature Medicine 2020). The 2nd CAR in the construct was a novel anti-CD20 CAR incorporating a human anti-CD20 scFv, plus 4-1BB and CD3ζ domains. This bicistronic CAR construct, designated Hu1928-Hu20BB-original, was encoded by the mouse stem cell virus-based splice-gag (MSGV1) gamma-retroviral vector.Repeated segments of identical DNA sequence can lead to recombination events when retroviral vectors are utilized. Recombination events driven by repeated identical nucleotide segments can occur during retroviral vector production in packaging cells. Recombination events can also occur in transduced T cells during reverse transcription when repeated segments of identical nucleotides on the same nucleotide strand anneal, which leads to strand switching by the reverse transcriptase enzyme and deletion of part of the sequence.We assessed T cells transduced with Hu1928-Hu20BB-original for evidence of recombination. Southern blots of DNA from T cells transduced with Hu1928-Hu20BB-original showed a dominant band of the expected CAR transgene length and no aberrant bands after 21 hours of exposure; however, with extended exposure of 8 days, an unexpected band indicating shorter transgene length became visible. This aberrant band was consistent with deletion of part of the transgene sequence due to recombination. To investigate possible recombination by a more sensitive method, we assessed RNA from transduced T cells by single-molecule real-time RNA sequencing (SMRT-RNAseq, PacBio). Deletions of Hu1928-Hu20BB-original RNA sequence between segments of identical sequence were detected by SMRT-RNAseq. These findings were consistent with recombination. The most common recombination events were between the CD8α hinge and transmembrane (HT) domains of the 2 CARs of Hu1928-Hu20BB-original. These HT domains shared regions of identical nucleic acid sequence. Less commonly, recombination events occurred between segments of identical sequence shared by the heavy-chain domains and the light-chain domains of the two CARs in the construct. The attached table summarizes deletions consistent with recombination in Hu1928-Hu20BB-original RNA from transduced T cells of one patient. Similar results were obtained in T cells of 3 other patients. We confirmed deletions consistent with recombination in CAR RNA transcripts by short-read RNA sequencing (Illumina). Digital droplet PCR analysis of DNA from transduced T cells showed that recombination events between the HT domains of Hu1928-Hu20BB-original were present in DNA of transduced T cells.We modified the DNA sequence of Hu1928-Hu20BB-original to reduce repeated segments of identical DNA sequence to form a new CAR designated Hu1928-Hu20BB-standard. When T cells transduced with Hu1928-Hu20BB-standard were studied by SMRT RNAseq, deletions of RNA sequence consistent with recombination were substantially reduced (Table).We modified Hu1928-Hu20BB-standard by lengthening the linker in the anti-CD20 scFv to form a construct designated Hu1928-Hu20BB-long. We compared Hu1928-Hu20BB-standard to Hu1928-Hu20BB-long in vitro, and both specifically recognized CD19 and CD20 in assays of degranulation, cytokine release, and proliferation. In vitro assays showed higher interleukin-2 release by T cells transduced with Hu1928-Hu20BB-long versus Hu1928-Hu20BB-standard in response to 3 different CD20+ target cells (P value range 0.001 to 0.036). We tested Hu1928-Hu20BB-long against tumors established in nod-scid common gamma-chain knockout (NSG) mice. Human T cells expressing Hu1928-Hu20BB-long eliminated tumors of the following tumor cell lines: st486 (CD19+, CD20+), st486-CD19-negative (CD20+, CD19 low to negative expression), and NALM6 (CD19+, CD20 low expression). One million Hu1928-Hu20BB-long T cells per mouse eradicated st486 tumors. These optimized anti-CD19/CD20 bicistronic CAR constructs are promising for clinical treatment of B-cell malignancies. [Display omitted] DisclosuresLam: Kite, a Gilead Company: Patents & Royalties. Kochenderfer: Bristol Myers Squibb: Research Funding; Kite, a Gilead Company: Patents & Royalties: on anti-CD19 CARs, Research Funding.

Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

Epigenetic memories and the evolution of infectious diseases

Genes with identical DNA sequence may show differential expression because of epigenetic marks. Where epigenetic marks respond to past conditions, they represent a form of “memory”. Despite their medical relevance, the impact of memories on the evolution of infectious diseases has rarely been considered. Here we explore the evolution of virulence in pathogens that carry memories of the sex of their previous host. We show that this form of memory provides information about the sex of present and future hosts when the sexes differ in their pathogen’s transmission pattern. Memories of past hosts enable the evolution of greater virulence in infections originating from one sex and infections transmitted across sexes. Thus, our results account for patterns of virulence that have, to date, defied medical explanation. In particular, it has been observed that girls infected by boys (or boys infected by girls) are more likely to die from measles, chickenpox and polio than girls infected by girls (or boys infected by boys). We also evaluate epigenetic therapies that tamper with the memories of infecting pathogens. More broadly, our findings imply that pathogens can be selected to carry memories of past environments other than sex. This identifies new directions in personalised medicine.