Comparative DNA Sequence Analysis Research Articles

Abstract C045: Investigating molecular and genetic contributions to racial disparities in prostate cancer by integrating multi-omic datasets available in public

Abstract African-American males are disproportionately affected by prostate cancer in comparison to other racial and ethnic groups. African-American males have increased risk for the disease, experience higher incidence and mortality rates, and present higher grade and stage prostate tumors than Caucasian men. To determine molecular mechanisms beyond racial disparities in prostate cancer, molecular and genetic profiles of prostate tumor samples from different ethnic groups are developed. For example, DNA sequencing in the form of target-based array or whole exome and genome sequencing revealed SNPs, genetic mutations, deletions, and amplifications linked to prostate cancer subgroups. Comparative analysis of DNA sequences of prostate cancer cells have pinpointed areas of interest that suggest race specific mutations. Moreover, differentially expressed genes between prostate tumor tissue samples from African and Caucasian ancestries are identified using RNA sequencing and microarray-based methods. Abnormal levels of gene expression are measured by the levels of mRNA. Furthermore, epigenetic modifications, such as DNA methylation levels were examined, identifying CpG sites that are differentially methylated among ethnic groups. To comprehensively examine molecular and genetic contributions to racial disparities in prostate cancer, we compiled over 200 published studies that profiled genome, epigenome, and transcriptome in prostate tumor tissues from diverse ethnic groups. Specifically, we categorized studies according to their method and organized the identified features that offer insight into racial disparities in prostate cancer. For instance, we reviewed over 100 studies that identify reproducible genetic alterations found in prostate cancer patients from African ancestry from DNA sequencing experiments. We also reviewed over 50 studies that utilized RNA sequencing to measure gene expression levels in prostate tumor tissues from African-American male samples, identifying genes and signaling pathways abnormally dysregulated in the disease. In addition, we reviewed over 25 studies quantifying epigenetic modifications to determine key and reproducible epigenetic alterations linked to aggressiveness of the disease from African American. Subsequently, we identified socioeconomic factors that are associated with molecular features contributing to the increased risk for prostate cancer in African-American men. Overall, we evaluated multiple molecular and genetic features that contribute to racial disparities in prostate cancer. This may provide researchers with valuable background knowledge and perspective for identifying biomarkers for specific prostate cancer subgroups. Citation Format: Alexandria J. Hightower, Suhn K. Rhie, Sara M. Falzarano, Sarah Buxbaum. Investigating molecular and genetic contributions to racial disparities in prostate cancer by integrating multi-omic datasets available in public [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C045.

Assessment of Rhodolith Diversity in the Northwestern Gulf of Mexico Including the Description of Sporolithon gracile sp. nov. (Sporolithales, Rhodophyta), and Three New Species of Roseolithon (Hapalidiales, Rhodophyta)

In the past, non-geniculate coralline algae in the northwestern Gulf of Mexico have been identified based primarily on comparative morpho-anatomy. Recent studies employing DNA sequencing techniques combined with morpho-anatomical studies using SEM have revealed a wealth of previously undocumented diversity of rhodolith-forming non-geniculate coralline algae in the Corallinales, Hapalidiales and Sporolithales from mesophotic hard bank communities at 45-90 meters depth. Although many advances in the last decade have been made in clarifying species names and describing new species of corallines from offshore Louisiana and Texas, total diversity estimates are still incomplete and many species remain to be described. Collections from offshore Louisiana at Parker Bank in the newly expanded Flower Garden Banks National Marine Sanctuary yielded thin, finely branched rhodoliths. DNA sequence analyses of plastid-encoded psbA and rbcL loci, and nuclear-encoded LSU rDNA of these rhodolith-forming specimens revealed that some belong to an unnamed species of Sporolithon (Sporolithales) that we herein newly describe. Additionally, comparative DNA sequence analyses of rhodolith collections from Ewing Bank and other hard banks offshore Louisiana were conducted to assess rhodolith diversity in these mesophotic communities. The results revealed new reports of taxa for the region, including new rhodolith-forming species of Roseolithon (Hapalidiales) to be described herein as well. Our new biodiversity findings will be compared with historical studies from the NW Gulf of Mexico.

Differential Etv2 threshold requirement for endothelial and erythropoietic development.

SUMMARYEndothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

Molecular epidemiology of Aleutian mink disease virus from fecal swab of mink in northeast China

BackgroundAleutian mink disease parvovirus (AMDV) causes Aleutian mink disease (AMD), which is a serious infectious disease of mink. The aim of this study was to get a better understanding of the molecular epidemiology of AMDV in northeast China to control and prevent AMD from further spreading. This study for the first time isolated AMDV from fecal swab samples of mink in China.ResultsA total of 157/291 (54.0%) of the fecal swab samples were positive for AMDV. Of these, 23 AMDV positive samples were randomly selected for sequence alignment and phylogenetic analysis based on the acquired partial fragments of VP2 gene with the hypervariable region. Comparative DNA sequence analysis of 23 AMDV isolates with a reference nonpathogenic (AMDV-G) strain revealed 8.3% difference in partial VP2 nucleotide sequences. Amino acid alignment indicated the presence of several genetic variants, as well as one single amino acid residue deletion. The most concentrated area of variation was located in the hypervariable region of VP2 protein. According to phylogenetic analysis, the Chinese AMDV strains and the other reference AMDV strains from different countries clustered into three groups (clades A, B and C). Most of the newly sequenced strains were found to form a Chinese-specific group, which solely consisted of Chinese AMDV strains.ConclusionThese findings indicated that a high genetic diversity was found in Chinese AMDV strains and the virus distribution were not dependent on geographical origin. Both local and imported AMDV positive species were prevalent in the Chinese mink farming population. The genetic evidence of AMDV variety and epidemic isolates have importance in mink farming practice.

Two novel Rickettsia species of soft ticks in North Africa: ‘Candidatus Rickettsia africaseptentrionalis’ and ‘Candidatus Rickettsia mauretanica’

Rickettsia are obligate intracellular bacteria often reported from hard ticks but more rarely from soft ticks. In this study, we detected in Northern Africa two putatively novel Rickettsia species in soft tick species of the Ornithodoros erraticus complex: Ornithodoros occidentalis from Morocco, Ornithodoros erraticus from Algeria and Ornithodoros normandi from Tunisia. We characterized these two novel Rickettsia species on the basis of comparative DNA sequence analyses and phylogenetics of four genes (gltA, 16S rRNA, coxA and ompB). These Rickettsia, provisionally named ‘Candidatus Rickettsia africaseptentrionalis’ and ‘Candidatus Rickettsia mauretanica’, differed in nucleotide sequence from those of other Rickettsia species by 0.38–21.43 % depending on the gene examined. Phylogenetics further showed that the two novel Rickettsia species are closely related to each other and represent sister taxa to R. hoogstraalii, R. felis and R. asembonensis within the transitional Rickettsia group. While Ornithodoros host species of ‘Candidatus Rickettsia africaseptentrionalis’ and ‘Candidatus Rickettsia mauretanica’ are among the most common soft ticks to bite humans, their pathogenicity remains to be investigated.

DNA Sequencing Historical Lichen Specimens

Biological specimens in natural history collections worldwide are increasingly being used in biogeographical, environmental, and taxonomic studies. For their meaningful use, correct species identification is crucial. For example, clarifying if a species is new to science requires an overview of what has already been described. This includes comparisons with existing authoritative specimens (types). Most type specimens are rather old and their DNA expected to be degraded to various extents. Comparative DNA sequence analysis is in regular use in taxonomic research of today and is essential for identifying and delimiting species. In this study, we focus on lichenized fungi (lichens), in which many species groups are highly inconspicuous and impossible to identify to species based on morphology alone. Our aim was to test the non-mutually exclusive hypotheses that DNA quality of lichens depends on (1) time since collection, (2) taxonomic affinity, and/or (3) habitat/ecology. We included two species from each of four different lichen genera (i.e., Cladonia, Nephroma, Peltigera and Ramalina), each species pair with a different autecology. For each species, we included samples from approximately every 25 years from present to about 150 years back in time. We used a two-step PCR-based approach followed by sequencing on an Ion Torrent PGM to produce target sequences (mtSSU) of degraded DNA. We received satisfactory DNA sequence information for 54 of 56 specimens. We recovered full-length sequences for several more than 100-year-old specimens, including a 127-year-old specimen, and retrieved enough sequence information for species identification of a 150-year-old specimen. As expected, sequencing success was negatively correlated with age of the specimens. It also varied with taxonomic affinity. We found no significant correlation between sequencing success and habitat ecology of the investigated specimens. The herein tested Ion Torrent sequencing approach outperformed Sanger sequencing with regard to sequencing success and efficiency. We find the protocol used herein highly suitable for obtaining sequences from both young and old lichen specimens and discuss potential improvements to it.

Bacterial and fungal biodeterioration of discolored building paints in Lagos, Nigeria.

Microbial induced discolorations are an unsightly feature occurring on painted walls in Lagos, the commercial hub of Nigeria. Very few studies have been carried out conventionally about the microbial community structure of discolored painted walls in Nigeria therefore, knowledge of the true microbial diversity is elusive. To further our understanding of the phylogenetic diversity of representative microbial community on 40 discolored and three clean-looking buildings, a comparative DNA sequence analysis of 16S rDNA genes was undertaken. Following DNA extraction, portions of the rDNA genes were amplified by PCR and sequenced. Resulting sequences were compared with GenBank data base sequences. Fifteen unique fungal sequences and one bacterial sequence were obtained. Majority (37.50%) of rDNA sequences analyzed, represent the genus Meyerozyma of which the novel fungus Meyerozyma guilliermondii, which to our knowledge, has not yet been implicated in painted walls was detected. Clones from the discolored painted wall isolates also produced a data set in which 31.25% of sequences were related to Fusarium proliferatum and 6.25% were Pseudomonas aeruginosa. The remaining sequences clustered with members of the genera Candida (6.25%), Aspergillus (12.5%) and Cerrena (6.25%). The study provides reliable data on microbial communities on painted walls and information for paint biocide formulation.

The Investigation of Organelle Genomes of Extra Nuclear Sunflower Mutants with Variegated Phenotype

The comparative analysis of complete chloroplast and mitochondrial DNA sequences of the original inbred line 3629 and three extra nuclear mutant lines variegated-1, variegated-15, variegated-17, obtained by the N-nitroso-N-methylurea induced mutagenesis, was done. The studied mutant lines were presented two different phenotypes with diverse chlorophyll content: Pale/green mutant line variegated-17 with 1.8% relative (to 3629 line) chlorophyll content in mutant leaf tissue and yellow/green lines variegated-1, variegated-15 with 6.3% and 4.3% relative chlorophyll content, respectively. Each line had unique variation sites in chloroplast DNA. Among chloroplast SNP there were determined 7 nonsynonymous substitutions, which were located in psaA (variegated-1), petD, rpl36, ccsA (variegated-15), rps4, rpoA, rpoC1 (variegated-17). The SNP of variegated-17 line has resulted in the frameshift (rpoC1) and premature stop codon formations (rpoA, rpoC1). The whole genome sequencing of mitochondrial DNA mutant lines revealed no differences as compared with original line 3629. Summarizing current data and our previous research of variegated mutants we assume that pale/green variegated phenotype is associated with significant disturbance of RNA polymerase subunits genes, and the lesions in photosynthetic genes lead to yellow/green mutant phenotype.

Complete Chloroplast Genome of Medicinal Plant Lonicera japonica: Genome Rearrangement, Intron Gain and Loss, and Implications for Phylogenetic Studies.

The complete chloroplast (cp) genome of Lonicera japonica, a common ornamental and medicinal plant in North America and East Asia, was sequenced and analyzed. The length of the L. japonica cp genome is 155,078 bp, contains a pair of inverted repeat regions (IRa and IRb), of 23,774 bp each, as well as large (LSC, 88,858 bp) and small (SSC, 18,672 bp) single-copy regions. A total of 129 genes were identified in the cp genome, 16 of which were duplicated within the IR regions. Relative to other plant cp genomes, the L. japonica cp genome had a unique rearrangement between trnI-CAU and trnN-GUU. In L. japonica cpDNA, rps19, rpl2, and rpl23 move to the LSC region, from the IR region. The ycf1 pesudogene in the IR region is lost, and only one copy locates in the SSC region. Comparative cp DNA sequence analyses of L. japonica with other cp genomes reveal that the gene order, and the gene and intron contents, are slightly different. The introns in ycf2 and rps18 genes are found for the first time. Four genes (clpP, petB, petD, and rpl16) lost introns. However, its genome structure, GC content, and codon usage were similar to those of typical angiosperm cp genomes. All preferred synonymous codons were found to use codons ending with A/T. The AT-rich sequences were less abundant in the coding regions than in the non-coding ones. A phylogenetic analysis based on 71 protein-coding genes supported the idea that L. japonica is a sister of the Araliaceae species. This study identified unique characteristics of the L. japonica cp genome that contribute to our understanding of the cpDNA evolution. It offers valuable information for the phylogenetic and specific barcoding of this medicinal plant.

Approach to determine the diversity of Legionella species by nested PCR-DGGE in aquatic environments

In this study, we describe a nested PCR-DGGE strategy to detect Legionella communities from river water samples. The nearly full-length 16S rRNA gene was amplified using bacterial primer in the first step. After, the amplicons were employed as DNA templates in the second PCR using Legionella specific primer. The third round of gene amplification was conducted to gain PCR fragments apposite for DGGE analysis. Then the total numbers of amplified genes were observed in DGGE bands of products gained with primers specific for the diversity of Legionella species. The DGGE patterns are thus potential for a high-throughput preliminary determination of aquatic environmental Legionella species before sequencing. Comparative DNA sequence analysis of excised DGGE unique band patterns showed the identity of the Legionella community members, including a reference profile with two pathogenic species of Legionella strains. In addition, only members of Legionella pneumophila and uncultured Legionella sp. were detected. Development of three step nested PCR-DGGE tactic is seen as a useful method for studying the diversity of Legionella community. The method is rapid and provided sequence information for phylogenetic analysis.

Comparative analysis of chloroplast DNA sequences of Codonopsis lanceolata and Platycodon grandiflorus and application in development of molecular markers

Codonopsis lanceolata and Platycodon grandiflorus (order Asterales) originate from East Asia. Despite the high commercial availability of C. lanceolata and P. grandiflorus, limited genetic research has been performed on these plants. We applied a targeting enrichment method to detect genetic diversity in C. lanceolata and P. grandiflorus and recovered their chloroplast genomes from total DNA sequence data. Chloroplast DNAs (cpDNAs) were 61,154 bp (C. lanceolata) and 81,214 bp (P. grandiflorus) in length. Sixteen simple sequence repeats and 15 long repeat sequences were determined, which are useful as potential markers in both plant species. We surveyed the phylogenetic relationships with increased resolution in 14 plant species, including other 8 species from the order Asterales and 4 from the order Apiales. In addition, we demonstrated the availability of recovered chloroplast genomes through cpDNA marker development to determine the authenticity of food fraud at the DNA level of plant species.

Phylogenetic Analysis Using Protein Mass Spectrometry.

Through advances in molecular biology, comparative analysis of DNA sequences is currently the cornerstone in the study of molecular evolution and phylogenetics. Nevertheless, protein mass spectrometry offers some unique opportunities to enable phylogenetic analyses in organisms where DNA may be difficult or costly to obtain. To date, the methods of phylogenetic analysis using protein mass spectrometry can be classified into three categories: (1) de novo protein sequencing followed by classical phylogenetic reconstruction, (2) direct phylogenetic reconstruction using proteolytic peptide mass maps, and (3) mapping of mass spectral data onto classical phylogenetic trees. In this chapter, we provide a brief description of the three methods and the protocol for each method along with relevant tools and algorithms.

Procaryotic species and subspecies delineation using average nucleotide identity and gene order conservation

Two genome-based parameters, gene order conservation (GOC) and average nucleotide identity (ANI) were studied to define how accurately two species, or two subspecies could be distinguished on the basis of these parameters. The first examined species was Lactococcus lactis since its two subspecies, the subsp. lactis and the subsp. cremoris have complete genome sequences available. Comparative analysis of DNA sequences of two subspecies of Streptococcus equi, S. equi subsp. equi and S. equi subsp. zooepidemicus were performed. In addition, S. thermophilus and S. salivarius, which formerly were classified into one species, were also compared. The GOC values are 66% or higher if the strains belong to the same species and there is no difference in GOC data of strains belonging to either the same subspecies or different subspecies. However, if two different species are compared, the GOC value decreases below 50% enabling an accurate separation of two species. The GOC value of 66% corresponds to a DNA-DNA hybridisation value of 70%. Unlike GOC, ANI measurements allow distinction of subspecies. If the strains belong to the same subspecies, ANI values are higher than 96%. However, when strains from different subspecies are compared the values are between 88% and 90%. Therefore, the average nucleotide identity can be used to delineate the boundary between subspecies. All genomic data and results of phylogenetic analyses obtained here by comparing S. salivarius and S. thermophilus support their previous taxonomic classification that they are actually two subspecies and not two separate species.

Morphological and molecular confirmation of the occurrence of Pyropia tanegashimensis (Bangiales, Rhodophyta) from Palaui Is., Sta. Ana, Cagayan, Philippines

The known morphological features in identifying common species of foliose Bangiales (e.g., members of Pyropia and Porphyra), which are very few and often overlapping, are recently resolved using comparative analysis of DNA sequences and statistics. Records of foliose Bangiales in the Philippines were historically identified based on morphology. Considering the recent radical changes in the taxonomy among these rhodophytes and problems posed by morphology-based identification, taxonomic re-appraisal of Philippine foliose Bangiales based on critical morphological and molecular studies is needed. This study used plastid rbcL and mitochondrial COI-5P gene sequences to investigate the identity of foliose Bangiales collected in Palaui Is., Sta. Ana, Cagayan, Philippines. Observation of key phenotypes revealed the identity of the collected materials as Py. tanegashimensis. Resulting phylogenetic trees showed placement of our collected specimens into a highly supported Py. tanegashimensis clade from Japan and Brazil. Our molecular analysis also suggested that the Philippine Py. tanegashimensis includes endemic populations distinct from the introduced strain originally reported from Brazil.

Molecular evolution and functional characterisation of haplotypes of an important rubber biosynthesis gene in Hevea brasiliensis.

Hydroxy-methylglutaryl coenzyme-A synthase (HMGS) is a rate-limiting enzyme in the cytoplasmic isoprenoid biosynthesis pathway leading to natural rubber production in Hevea brasiliensis (rubber). Analysis of the structural variants of this gene is imperative to understand their functional significance in rubber biosynthesis so that they can be properly utilised for ongoing crop improvement programmes in Hevea. We report here allele richness and diversity of the HMGS gene in selected popular rubber clones. Haplotypes consisting of single nucleotide polymorphisms (SNPs) from the coding and non-coding regions with a high degree of heterozygosity were identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor to the generation of allelic diversity, rather than point mutations. The evolutionarily conserved nature of some SNPs was identified by comparative DNA sequence analysis of HMGS orthologues from diverse taxa, demonstrating the molecular evolution of rubber biosynthesis genes in general. In silico three-dimensional structural studies highlighting the structural positioning of non-synonymous SNPs from different HMGS haplotypes revealed that the ligand-binding site on the enzyme remains impervious to the reported sequence variations. In contrast, gene expression results indicated the possibility of association between specific haplotypes and HMGS expression in Hevea clones, which may have a downstream impact up to the level of rubber production. Moreover, haplotype diversity of the HMGS gene and its putative association with gene expression can be the basis for further genetic association studies in rubber. Furthermore, the data also show the role of SNPs in the evolution of candidate genes coding for functional traits in plants.

Boundaries of Morphological and Molecular Variation and the Distribution of a Miniaturized Froglet,Brachycephalus nodoterga(Anura: Brachycephalidae)

Abstract Most miniaturized froglets of the genus Brachycephalus occur in isolation in slopes of mountain ranges at elevations varying from 600 to 1,800 m in the Atlantic Forest of eastern Brazil. For organisms such as Bracycephalus with spatially discontinuous distributions, a fundamental task is to determine whether observed patterns of variation are consistent with geographic differentiation among allopatric populations within a single species or are suggestive of a potential species boundary. We address this problem by focusing on continental and island population samples potentially assignable to Brachycephalus nodoterga (Anura: Brachycephalidae) from the perspective of variation in qualitative and quantitative morphological traits and DNA sequences. Population samples from continental and island populations share color characteristics, qualitative traits, and multivariate patterns of variation and covariation in cranial metric traits. Comparative analysis of DNA sequences showed the magnitude of mole...

結石内部に真菌塊がみられた頰粘膜小唾液腺唾石症の1例

We describe a case of sialolithiasis of the buccal mucosa with a fungal mass in a calculus of the minor salivary gland. An 87-year-old man was referred to our hospital because of a small nodule in the right buccal mucosa. On examination, an elastic, hard, painless, movable nodule, measuring 3 × 3 mm and located under the right buccal mucosa, was detected. The overlying mucosal surface was intact. Radiographic examination revealed no abnormalities in the right buccal region. We resected the nodule with the patient under local anesthesia. Histological examination of the resected specimen revealed lightly eosinophilic material with lamellar calcification and colonies of yeast-like fungus in a duct of the minor salivary gland. Comparative 26S ribosomal DNA sequence analysis of paraffin sections identified the fungus as Candida parapsilosis. This case suggests a potential role of mycosis in the etiopathogenesis of sialolithiasis.

Origins of Wohlfahrtia magnifica in Italy based on the identification of mitochondrial cytochrome b gene haplotypes.

To identify the geographical origins of larvae of Wohlfahrtia magnifica (Diptera: Sarcophagidae) causing myiasis of sheep in Italy, comparative DNA sequence analysis of the mitochondrial cytochrome b gene was performed, based on gene fragments amplified by PCR from genomic DNA isolated from individual specimens. DNA extractions of 19 larvae from Lazio, Molise, Puglia, and Sicilia generated 17 readable sequences homologous to 2 haplotypes, either CB_magn01 or CB_magn02; DNA extracts from 4 adult flies from Calabria (reared from larvae) produced 4 readable sequences belonging to the haplotype CB_magn01. The two haplotypes found represent both the East and West phylogenetic lineages of W. magnifica, which is consistent with the species' arrival from central/southeast Europe (East lineage) and/or from southwest Europe/northwest Africa (West lineage). This is the first report of the sympatric occurrence of the two lineages, which could have resulted from natural or human-assisted dispersal. Polymorphic nuclear loci will have to be characterized in order to explain the origins and lack of mitochondrial haplotype diversity of this pest in Italy, where it poses increasing veterinary problems.

Comparative analysis of DNA sequences of regions of X-chromosome attachment to the nuclear envelope of nurse cells Anopheles messeae Fall.

Polytene chromosomes of ovarian nurse cells of Anopheles mosquitoes form strong contacts with the nuclear envelope. The presence of contacts, their position at nurse cell chromosomes, and their morphological features are species-specific in malaria mosquitoes. It is important to determine the nature of these interspecies differences in the nuclear architecture, both to understand the function of the nucleus and to assess the role of the spatial organization of chromosomes in evolution. Using dot-blot hybridization, we compared DNA sequences of the clone library from the X-chromosome attachment region to the nuclear envelope of ovarian nurse cells of Anopheles messeae with DNA-probes: (1) of the X-chromosome attachment region of An. atroparvus, (2) of the 3R chromosome attachment region ofAn. messeae, and (3) of the chromosome 2 pericentromeric region of An. messeae, without expressed contacts with the nuclear envelope. It has been shown that the chromosome attachment regions have a significantly higher number of homologous DNA sequences as compared with the pericentromeric region of chromosome 2. Sequences that are common for attachment regions are largely potentially able to participate in the formation of chromatin loop domains and to interact with some nucleus frameworks, according to the analysis in the ChrClass program. The obtained results support the important role of DNA in the formation of strong chromosomal attachments to the nuclear envelope in nurse cells of Anopheles mosquitoes.

Two Phages, phiIPLA-RODI and phiIPLA-C1C, Lyse Mono- and Dual-Species Staphylococcal Biofilms.

Phage therapy is a promising option for fighting against staphylococcal infections. Two lytic phages, vB_SauM_phiIPLA-RODI (phiIPLA-RODI) and vB_SepM_phiIPLA-C1C (phiIPLA-C1C), belonging to the Myoviridae family and exhibiting wide host ranges, were characterized in this study. The complete genome sequences comprised 142,348 bp and 140,961 bp and contained 213 and 203 open reading frames, respectively. The gene organization was typical of Spounavirinae members, with long direct terminal repeats (LTRs), genes grouped into modules not clearly separated from each other, and several group I introns. In addition, four genes encoding tRNAs were identified in phiIPLA-RODI. Comparative DNA sequence analysis showed high similarities with two phages, GH15 and 676Z, belonging to the Twort-like virus genus (nucleotide identities of >84%); for phiIPLA-C1C, a high similarity with phage phiIBB-SEP1 was observed (identity of 80%). Challenge assays of phages phiIPLA-RODI and phiIPLA-C1C against planktonic staphylococcal cells confirmed their lytic ability, as they were able to remove 5 log units in 8 h. Exposure of biofilms to phages phiIPLA-RODI and phiIPLA-C1C reduced the amount of adhered bacteria to about 2 log units in both monospecies and dual-species biofilms, but phiIPLA-RODI turned out to be as effective as the mixture of both phages. Moreover, the frequencies of bacteriophage-insensitive mutants (BIMs) of Staphylococcus aureus and S. epidermidis with resistance to phiIPLA-RODI and phiIPLA-C1C were low, at 4.05 × 10(-7) ± 2.34 × 10(-9) and 1.1 × 10(-7) ± 2.08 × 10(-9), respectively. Overall, a generally reduced fitness in the absence of phages was observed for BIMs, which showed a restored phage-sensitive phenotype in a few generations. These results confirm that lytic bacteriophages can be efficient biofilm-disrupting agents, supporting their potential as antimicrobials against staphylococcal infections.