Restriction Enzyme Research Articles

Evolution of a small phage protein overcomes antiphage defense in Enterococcus faecalis.

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of new antibiotics needed to combat these infections remains stagnant. MDR enterococci, which are a common cause of hospital-acquired infections, are emerging as one of the major contributors to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which entails the use of lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of phage resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from vancomycin-resistant Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) and we show that this enzyme is sufficient to restrict the replication of lytic phage phi47 in E. faecalis . We further find that phi47 can evolve to overcome restriction by acquiring a missense mutation in a novel TIV-RE inhibitor protein encoded by many enterococcal phages. We show that this inhibitor, which we have named type IV restriction inhibiting factor A ( tifA ), directly binds to and inactivates diverse TIV-REs. Overall, our findings significantly advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages can evolve to overcome antiphage defense systems.

The Relationship Between Body Weight and Growth Hormone Gene Polymorphism in Indigenous Turkeys of Nigeria

The candidate gene approach is a valuable tool for improving breeding programs, particularly in poultry, by identifying traits for enhanced production performance. This study investigates the growth hormone (GH) gene as a potential determinant of body weight in Nigerian indigenous turkeys. DNA was extracted from 60 twelve-week-old turkeys using blood samples, and a 466bp DNA fragment was amplified via PCR with primers designed from chicken sequences. The PCR product underwent Msp1 restriction enzyme digestion and was visualized on a 1.5% agarose gel. Genotypes associated with body weight were assessed using a General Linear Model. The GH/Msp1 locus revealed Two alleles (A and G) and three genotypes (AA, AG, and GG), with allele G being predominant. Genotype AG had the highest frequency (0.55), followed by AA (0.42), and GG had the lowest frequency (0.03). However, the least square means analysis showed that the polymorphism in the GH gene of the studied population was not significantly associated with body weight (p> 0.05). Nonetheless, the polymorphic nature of the GH gene in Nigerian turkeys suggests potential for further exploration in quantitative traits.

T-RFLP biomolecular indicator for partial nitritation under saline conditions and machine learning application

This study introduces the smart development of a cost-effective biomolecular indicator using terminal restriction fragment length polymorphism (T-RFLP) based on the 16S rRNA gene database obtained from operating the partial nitritation (PN) process under saline conditions. As a result of next-generation sequencing (NGS) on the 16S rRNA gene, ammonia-oxidizing bacteria (AOB) of Nitrosomonas sp. OTU0 (N. OTU0) favored higher salinity of 20–35 g-NaCl/L, while Nitrosomonas sp. OTU8 (N. OTU8) and Nitrosomonas sp. OTU11 (N. OTU11) were predominant under 20 g-NaCl/L. The T-RFLP system with the restriction enzymes of TaiI and FnuDII, which was thoroughly simulated based on the NGS data of the 16S rRNA gene, effectively differentiates N. OTU0 against N. OTU8 and N. OTU11. In addition, together with AOB’s signals, the signals of nitrite-oxidizing and heterotrophic bacteria were obtained as biomolecular indicators of successful PN under saline conditions. These findings suggest that T-RFLP can serve as an economically viable monitoring approach for the sensitive PN process under saline conditions to achieve more sustainable nitrogen removal. For further application, the designated T-RFLP signals of this study were applied to the regression of ammonia conversion rate through machine learning modeling. The regression showed R2 values of 0.9446 and 0.9458 for the training and test sets, respectively. The contribution of T-RFLP for the regression was quantified by a shapley additive explanation (SHAP).

First report of the new Neopestalotiopsis species causing strawberry leaf spot and fruit rot in Georgia.

In April 2023, symptomatic strawberry (Fragaria × ananassa) plants (cv. 'Camarosa' and 'Florida Brilliance') were observed at a commercial farm in Worth County, GA (USA). Symptoms included foliar, irregularly distributed, and different-sized spots (dark brown with light brown centers) and dark brown V-shaped necrotic areas starting at the leaf edge. By the time of sample collection, ~50% incidence was reported in the field. Leaf samples were collected and shipped overnight to the laboratory. Black acervuli were observed readily on old necrotic foliar lesions. Conidial morphology was consistent with that observed with Neopestalotiopsis species (Maharachchikumbura et al. 2014). Conidia were ellipsoid to fusiform, five-celled, with three light brown median cells and one hyaline apical and basal cell. Apical cells had two-to-four flexuous appendages, and the basal cell had one non-flexuous appendage (Fig 1). The average (n = 20) conidia length, not including the appendages, was 26.6 µm (SD: 2.8), and width was 6.3 µm (SD: 0.94). Fungal isolation was conducted on acidified PDA and incubated at 25°C for 6 days. Dense, white mycelia were observed on the upper plate surface, while a pale pink/orange coloration was observed on the underside (Fig 1). Black acervuli formed on the surface of the white mycelial mat. Six isolates were purified and selected to confirm the species identity. DNA was extracted from 6-day-old cultures and PCR was conducted following Kaur et al. (2023). Amplified DNA was digested with the restriction enzyme BsaWI and two bands were clearly visualized (~130 and ~290 bp), along with a faint band of 20-bp (Fig 2). Four of the six isolates were selected for sequencing of the β-tubulin gene. BLAST queries using the consensus sequence showed that all isolates had 100% identity to strain N21002 from Florida (FL), characterized as Neopestalotiopsis sp. (Kaur et al. 2023). One representative isolate (AJ07-2023) was deposited in GenBank (accession No. PP316103). Pathogenicity tests were performed on 27-day-old transplants of Sensation 'Florida127' provided by Natalia Peres from the UF. Plants were grown in 10.5 cm pots in the greenhouse. Isolate AJ07-2023 was grown on PDA for 30 days at 25°C, and the spore suspension was adjusted at 106 spore/ml. Five strawberry plants were sprayed with 5 ml of inoculum using a Preval sprayer with a CO2 canister, and 5 plants were sprayed with sterile distilled water. Plants were placed in a growth chamber for 6 days and covered with plastic bags after the sixth day to maintain ~85% relative humidity and 25°C. Foliar symptoms, including dark-brown circular lesions occurring towards the edge of leaves with light-brown center and light-yellow halo, developed 13 days after inoculation. No symptoms were observed on control plants. Neopestalotiopsis sp. was reisolated from inoculated plants as described above. Colony, conidial morphology, and PCR results were consistent with the original isolates. Neopestalotiopsis disease has been reported on strawberry in FL (Baggio et al. 2021), OH (Rotondo et al. 2022) and IN (Guan et al. 2023). Although the disease has been observed sporadically in GA since 2020 (Brannen, personal communication), to our knowledge, this is the first official report of the new Neopestalotiopsis sp. in GA. It has been reported that this new strain is more aggressive on fruits and leaves than other Neopestalotiopsis spp. (Baggio et al. 2021), therefore, accurate identification is critical for proper management.

Polymorphism of Cyt-b Gene in Several Indonesian Cattle Using PCR-RFLP Method

The cytochrome b (Cyt-b) gene is one of the genes that is located in the mitochondrial DNA. Variations in the Cyt-b gene can be used to compare different animal species to investigate the origin of certain animal species. This study aimed to assess the genetic diversity of Indonesian local cattle breeds, including Bali cattle as an Indonesian native cattle breed and Banteng as the wild type of Bali cattle, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A total of 336 individual DNA samples from Indonesian cattle breeds were analyzed in this study. The RFLP method using three restriction enzymes, i.e., HinfI, HaeIII, or XbaI, was used to identify the variation of the Cyt-b gene. The Cyt-b gene was polymorphic based on the PCR-RFLP method. There were six alleles of the Cyt-b gene found in this study, i.e., A and B allele (HinfI), C and D allele (HaeIII), X and Y allele (XbaI). All alleles can be found in Pasundan, Madura, and PO cattle. Pesisir cattle have the highest allele frequency D. The UPGMA results showed three clusters of Indonesian native cattle in this study. Cluster 1 consists of Pasundan, Banteng, and Bali cattle. Cluster 2 consists of Madura, PO, and SO cattle. Pesisir was separated from other cattle in cluster 3. The X allele could become an indicator to distinguish Banteng and Bali cattle.

CRISPR/Cas13a-based supersensitive circulating tumor DNA assay for detecting EGFR mutations in plasma

Despite recent technological advancements in cell tumor DNA (ctDNA) mutation detection, challenges persist in identifying low-frequency mutations due to inadequate sensitivity and coverage of current procedures. Herein, we introduce a super-sensitivity and specificity technique for detecting ctDNA mutations, named HiCASE. The method utilizes PCR-based CRISPR, coupled with the restriction enzyme. In this work, HiCASE focuses on testing a series of EGFR mutations to provide enhanced detection technology for non–small cell lung cancer (NSCLC), enabling a detection sensitivity of 0.01% with 40 ng cell free DNA standard. When applied to a panel of 140 plasma samples from 120 NSCLC patients, HiCASE exhibits 88.1% clinical sensitivity and 100% specificity with 40 μL of plasma, higher than ddPCR and Super-ARMS assay. In addition, HiCASE can also clearly distinguish T790M/C797S mutations in different positions at a 1% variant allele frequency, offering valuable guidance for drug utilization. Indeed, the established HiCASE assay shows potential for clinical applications.

Association of deletion polymorphism rs10573247 in the HMGA2 gene with the risk of breast cancer: bioinformatic and experimental analyses

BackgroundThe high mobility group A2 (HMGA2) gene is expressed extensively during early embryonic development but is inactivated in adulthood, and it is also reactivated in various benign and malignant tumors, including breast cancer. We first assessed the potential functional significance of the unstudied deletion polymorphism rs10573247 at the 3′UTR of HMGA2 on miRNA binding using bioinformatic tools, and subsequently, the association between this polymorphism and breast cancer susceptibility was investigated.Materials and methodsWe applied the RNAhybrid tool to predict the functional effects of polymorphism rs10573247 located within the 3’ UTR of the HMGA2 gene on miRNA binding. Then, following DNA extraction, 141 breast cancer patients and 123 healthy controls were genotyped for polymorphism rs10573247 using RFLP-PCR with the restriction enzyme Eam1104I.ResultsOur bioinformatic data have shown that polymorphism rs10573247 is located in the region that serves as a potential target site for eight miRNAs binding. Among them, miR-3125 exhibited decreased binding affinity for the allele delTT (MFE = -21.8) when compared to the allele TT (MFE = -23.9), but miR-4476 increased binding affinity for the allele delTT (MFE = -22.4) compared to the allele TT (MFE = -22.2). In addition, our results showed that the genotype TT/delTT (p = 0.005) and the genotype delTT/delTT (p = 0.029) were significantly associated with an increased risk of developing breast cancer compared to the genotype TT/TT using RFLP-PCR.Discussion and ConclusionOur findings suggest that polymorphism rs10573247 may contribute to the risk of breast cancer through the functional effect of this polymorphism on miRNA binding.

Identification of guaiacol producing <i>Alicyclobacillus</i> recovered from commercial orange juices distributed in Greek markets

The objective of this work was the isolation and characterization of Alicyclobacillus acidoterrestris strains from orange juice. In all, 72 isolates were recovered from commercial orange juice bought from the Greek market. The isolates were subjected to polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) to discriminate them at strain level and A. acidoterrestris types I and II. The restriction patterns of 16S rRNA gene revealed that isolates were categorized in different groups depending on the restriction endonucleases. Moreover, the digestion of the part of vdc operon gene with HphI restriction enzyme successfully differentiated between A. acidoterrestris types I and II. In conclusion, RFLP succeeded in differentiating A. acidoterrestris isolates at strain and type levels.

Development and clinical evaluation of a real-time multiple cross displacement amplification assay for rapid and sensitive detection of Mycobacterium tuberculosis

Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the Mycobacterium tuberculosis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 min) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.

#191 An unusual cause of hypocalcemia in a young patient

Abstract Background and Aims A 33-year-old man visited the Nephrology Department outpatient clinic with abnormal laboratory findings: his serum calcium level was 5.9 mg/dL in a recent health examination. He denied any underlying disease and was on no medications. He complained of a tingling sensation in his hands and of eyelid twitching. On physical examination, he showed Chvostek's sign. His laboratory findings including serum calcium level are shown in Table 1. He did not have any morphological abnormalities, such as a round face, brachydactyly, or dental anomalies. Method Since he showed hypocalcemia, normal 25-hydroxyvitamin D, and elevated parathyroid hormone (PTH) levels, the presence of PTH resistance was presumed. Under strong suspicion of pseudohypoparathyroidism (PHP) in this patient, DNA sequence analysis and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of GNAS and STX16, genetic regions that are often affected by mutations or imprinting changes in PHP, was performed. No pathogenic mutations in GNAS and STX16 were detected by sequencing. Results Results of MS-MLPA of the GNAS locus demonstrated abnormal methylation patterns in the patient after HhaI restriction enzyme treatment. The methylation ratio showed a gain of methylation in the NESP55 region and loss of methylation in the AS, XL, and A/B differentially methylated regions (DMRs) in the patient as compared to a healthy control (Fig. 1). The patient was diagnosed with PHP type 1B (PHP1B) due to the methylation abnormalities in multiple DMRs. His parents both showed normal serum calcium levels; thus, no further genetic testing was conducted. He is on outpatient follow-up with adequate calcium replacement and no specific symptoms. Conclusion PHP is a rare disorder of unknown prevalence. It is characterized by hypocalcemia, hyperphosphatemia, and increased PTH levels. PHP1B is a subtype of PHP characterized by isolated renal PTH resistance, usually without physical abnormalities such as Albright hereditary osteodystrophy or other endocrine abnormalities. PHP1B can be sporadic or familial. The genetic characteristics of most sporadic PHP1B remains unclear, but GNAS imprinting abnormalities and hypomethylation at the A/B DMRs have been reported. Symptoms of PHP1B usually begin in childhood due to low calcium levels and may include numbness, seizures, tetany, cataracts, and dental problems. However, as in our case, specific symptoms may not occur until adulthood. Therefore, when evaluating the cause of hypocalcemia, it is necessary to differentiate PHP1B, even in adults, and particularly in cases with hyperphosphatemia and high PTH levels.

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity.

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

Development and validation of a functional molecular marker for a CaAPRR2-like gene that controls green fruit colour in pepper

Fruit colour is a crucial factor influencing both the marketability and quality of pepper (Capsicum annuum), particularly ornamental varieties. Fruit colour is a complex multigenic trait in plant species. Previously, the Arabidopsis pseudo-response regulator 2 (APRR2) gene, one of the regulators that control fruit chlorophyll content and chloroplast development, thereby influencing fruit colour at the green immature stage in tomato (Solanum lycopersicum), cucumber (Cucumis sativus) and pepper, was reported. Functional molecular markers associated with this homologous gene in pepper could be employed to discern fruit colour at the seedling stage, thus improving the efficiency of green/white-fruited pepper breeding. In this study, a derived cleaved amplified polymorphic sequences (dCAPS) marker was developed based on the mutation site in the sequence of a CaAPRR2-like gene that was cloned from the inbred line B1-2, which exhibits milky white fruit at the green immature stage. The marker was subsequently validated in a CaAPRR2-like progeny segregation population. After digestion with the restriction endonuclease TaqI, the amplification product exhibited evident polymorphic bands, enabling it to distinguish between peppers with milky white and green fruit colours. The developed molecular marker displayed remarkable stability and repeatability, thus offering a straightforward and effective tool for enhancing fruit colour in pepper breeding programs.

Molecular Identification of Morus ssp. in Duhok Using Nuclear ITS Region and Chloroplast Matk Gene

Since Mulberries (Morus)is a tree species with a considerable plant variety. Molecular techniques are methods used to distinguish between species accurately, easily and quickly. This study examines a Molecular method for distinguishing different Morus species in the Duhok - Kurdistan region/ Iraq. The method is based on the use of four techniques: matK gene, the ITS region, PCR-RFLP, and SRAP markers. Twelve Morus species have been selected for this study from different region of Duhok. The ITS region's PCR result was 700 bp, but the matK gene's PCR produce was 900 bp. The same restriction site was found for all utilized cultivars when the 700bp of ITS fragment was used for PCR-RFLP with two restriction enzymes, RsaI GT/AC and HaeIII GG/CC. This study also used six combinations of SRAP markers to aid in grouping and identifying genetic similarities. The results of PCR-RFLP demonstrated an insufficient link between Morus physical appearance and genetic traits, but differences across studied cultivars could be identified using SRAP markers. Furthermore, this study demonstrated the possibility of DNA barcoding Morus cultivars, as well as additional sequence analysis and the identification of probable SNP between cultivars.

Characterize Type of Splicing Languages via Directed Splicing Graph

A splicing system is a formal characterization of the ability to generate certain enzymatic activities acting on deoxyribonucleic acid (DNA) molecules. In this paper, the results from Laun’s experiment are used in characterizing the type of splicing languages. In the experiments, two initial strings are involved with different features on the selected restriction enzymes. Case I and Case II discussed in this paper show that the splicing languages obtained from these experiments are in adult and limit languages. Nevertheless, the result obtained in this paper is more precise in showing the type of splicing languages which is beyond adult and limit languages when presented via a directed splicing graph. The features of the restriction enzyme that affect the formation of active persistent language are investigated based on the results proposed by Yusof.

Learning Micro-C from Hi-C with diffusion models.

In the last few years, Micro-C has shown itself as an improved alternative to Hi-C. It replaced the restriction enzymes in Hi-C assays with micrococcal nuclease (MNase), resulting in capturing nucleosome resolution chromatin interactions. The signal-to-noise improvement of Micro-C allows it to detect more chromatin loops than high-resolution Hi-C. However, compared with massive Hi-C datasets available in the literature, there are only a limited number of Micro-C datasets. To take full advantage of these Hi-C datasets, we present HiC2MicroC, a computational method learning and then predicting Micro-C from Hi-C based on the denoising diffusion probabilistic models (DDPM). We trained our DDPM and other regression models in human foreskin fibroblast (HFFc6) cell line and evaluated these methods in six different cell types at 5-kb and 1-kb resolution. Our evaluations demonstrate that both HiC2MicroC and regression methods can markedly improve Hi-C towards Micro-C, and our DDPM-based HiC2MicroC outperforms regression in various terms. First, HiC2MicroC successfully recovers most of the Micro-C loops even those not detected in Hi-C maps. Second, a majority of the HiC2MicroC-recovered loops anchor CTCF binding sites in a convergent orientation. Third, HiC2MicroC loops share genomic and epigenetic properties with Micro-C loops, including linking promoters and enhancers, and their anchors are enriched for structural proteins (CTCF and cohesin) and histone modifications. Lastly, we find our recovered loops are also consistent with the loops identified from promoter capture Micro-C (PCMicro-C) and Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET). Overall, HiC2MicroC is an effective tool for further studying Hi-C data with Micro-C as a template. HiC2MicroC is publicly available at https://github.com/zwang-bioinformatics/HiC2MicroC/.

A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction.

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly, vector construction becomes relatively simple and efficient. However, when the full-length genome of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cannot be easily amplified by a single polymerase chain reaction (PCR) from cDNA, or it is difficult to acquire a full-length gene expression vector by homologous recombination of multiple inserts in vitro, the current Gibson Assembly technique fails to achieve this goal. Consequently, we aimed to divide the PRRSV genome into several fragments and introduce appropriate restriction sites into the reverse primer for obtaining PCR-amplified fragments. After joining the previous DNA fragment into the vector by homologous recombination technology, the new vector acquired the restriction enzyme cleavage site. Thus, we can linearize the vector by using the newly added enzyme cleavage site and introduce the next DNA fragment downstream of the upstream DNA fragment. The introduced restriction enzyme cleavage site at the 3' end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3' end of the downstream DNA fragment. In this way, we can join DNA fragments to the vector one by one. This method is applicable to successfully construct the PRRSV expression vector and is an effective method for assembling a large number of fragments into the expression vector.

Identification and genotyping of Chikungunya virus using reverse transcription polymerase chain reaction and restriction fragment length polymorphism methods

INTRODUCTION. Chikungunya virus (CHIKV) genotyping involves sequencing fractions of genes encoding E1, E2, nsP1 proteins or the entire genome of the virus. Available reagent kits or polymerase chain reaction protocols cannot be used for CHIKV genotyping, and nucleic acid sequencing requires expensive equipment and materials, which are not always available. Therefore, it seems promising to use a simpler and more cost-effective restriction fragment length polymorphism (RFLP) method, which has not previously been used for CHIKV genotyping.AIM. This study aimed to investigate the possibility of using reverse transcription polymerase chain reaction (RT-PCR) and RFLP for CHIKV identification and genotyping.MATERIALS AND METHODS. The experimental study used RNA from CHIKV strains of four genotypes, including the Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL). The study used RT-PCR followed by DNA restriction and restriction fragment length analysis.RESULTS. The nsP2 gene fragment of 648 bp in length (positions 3806 to 4453) contains recognition sites for the restriction endonucleases PspEI, PvuII, and DraI. The presence or absence of these sites generates a different combination specific to each of the four CHIKV genotypes. The authors designed primers for amplification of the selected gene region and performed RT-PCR and RFLP.CONCLUSIONS. The RFLP method can be used for rapid CHIKV identification and genotyping. The method provides results within a few hours and does not require high-tech equipment.

Readsynth: short-read simulation for consideration of composition-biases in reduced metagenome sequencing approaches

BackgroundThe application of reduced metagenomic sequencing approaches holds promise as a middle ground between targeted amplicon sequencing and whole metagenome sequencing approaches but has not been widely adopted as a technique. A major barrier to adoption is the lack of read simulation software built to handle characteristic features of these novel approaches. Reduced metagenomic sequencing (RMS) produces unique patterns of fragmentation per genome that are sensitive to restriction enzyme choice, and the non-uniform size selection of these fragments may introduce novel challenges to taxonomic assignment as well as relative abundance estimates.ResultsThrough the development and application of simulation software, readsynth, we compare simulated metagenomic sequencing libraries with existing RMS data to assess the influence of multiple library preparation and sequencing steps on downstream analytical results. Based on read depth per position, readsynth achieved 0.79 Pearson’s correlation and 0.94 Spearman’s correlation to these benchmarks. Application of a novel estimation approach, fixed length taxonomic ratios, improved quantification accuracy of simulated human gut microbial communities when compared to estimates of mean or median coverage.ConclusionsWe investigate the possible strengths and weaknesses of applying the RMS technique to profiling microbial communities via simulations with readsynth. The choice of restriction enzymes and size selection steps in library prep are non-trivial decisions that bias downstream profiling and quantification. The simulations investigated in this study illustrate the possible limits of preparing metagenomic libraries with a reduced representation sequencing approach, but also allow for the development of strategies for producing and handling the sequence data produced by this promising application.

Phosphorus-Nitrogen compounds part 74. Syntheses of (p-dimethylamino)benzyl-spiro-(N/N)-cyclotriphosphazenes: Structural characterizations, bioactivity studies, DFT calculations and reactivity parameters

Reactions of hexachlorocyclotriphosphazene (HCCP, N3P3Cl6), with equimolar amounts of N-methyl-N'-(p-dimethylaminobenzyl)-1,2-diaminoethane (1), N-ethyl-N'-(p-dimethyl-aminobenzyl)-1,2-diaminoethane (2), and N-methyl-N'-(p-dimethylaminobenzyl)-1,3-diami-nopropane (3) produced the corresponding tetrachloro-(p-dimethylaminobenzyl)spiro-(N/N)-cyclotriphosphazenes (4, 5, and 6), regioselectively. Tetrapyrrolidino (4a-6a), tetrapiperidino (4b-6b), tetramorpholinophosphazenes (4c and 6c), dichlorodimorpholinophosphazene (5c) and tetra-1,4-dioxa-8-azaspiro[4,5]decanophosphazenes (DASD) (4d-6d) were synthesized from Cl-substitution reactions of 4, 5 and 6 with excess pyrrolidine, piperidine, morpholine, and DASD, respectively. Their structures were elucidated by MS, FTIR, NMR and single crystal X-ray diffraction (for 4, 4b and 6a) techniques. Hirshfeld surface (HS) analyzes of 4, 4b, and 6a were performed to visualize intermolecular interactions in the crystals. In addition, according to the antimicrobial activity test results, compound 6b shows a better inhibitory effect on S. aureus ATCC 25923 G(+) (MİC: 78.1 µM) and C. tropicalis Y-12968 (MİC: 19.54 µM) microorganisms. The interactions of cyclotriphosphazenes with 5′-G/GATCC-3′ and 5′-A/AGCTT-3′ sequences of DNA were evaluated by examining BamHI and HindIII restriction enzyme digestion. The quantum chemical calculations of 4, 4b, and 6a were performed using DFT, and their experimental parameters were compared with the theoretical ones. Natural bond orbitals (NBOs) were determined and HOMO-LUMO energy gaps (Eg) were calculated as 3.55, 5.23, and 5.24 eV for 4, 4b, and 6a, respectively.

Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus–host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.