Explore the vast range of titles available.

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants.

Marine actinomycetes are a potential source of a wide variety of bioactive natural products. In this work, seven pretreatments, three selective isolation media, and five artificial seawater concentrations were used to isolate actinomycetes from the sediments collected from Yellow Sea, China. Statistical analysis showed that only the isolation medium strongly affected the total and bioactive numbers of actinomycete isolates. A total of 613 actinobacterial strains were isolated and screened for antimicrobial activities; 154 isolates showed activity against at least one of nine test drug-resistant microorganisms. Eighty-nine representatives with strong antimicrobial activity were identified phylogenetically based on 16S rRNA gene sequencing, which were assigned to five different actinomycete genera Streptomyces, Kocuria, Saccharomonospora, Micromonospora, and Nocardiopsis. Using PCR-based screening for six biosynthetic genes of secondary metabolites, all 45 isolates with acute activity have at least one biosynthetic gene, 28.8 % of which possess more than three biosynthetic genes. As a case, strain SMA-1 was selected for antimicrobial natural product discovery. Three diketopiperazine dimers including a new compound iso-naseseazine B (1) and two known compounds naseseazine B (2) and aspergilazine A (3) were isolated by bioassay-guided separation. These results suggested that actinomycetes from marine sediments are a potential resource of novel secondary metabolites and drugs.

Pediococcus pentosaceus NCDC 273, a dairy strain was screened for production of an YGNGV motif containing antimicrobial peptide (AMP). In microbiological and PCR based screening, it was shown that P. pentosaceus NCDC273 produces an AMP i.e Pediocin 273, encoded by pedA gene. The molecular weight of pediocin was determined about 5.19 kDa. The purification yield was 438% and the specific activity was increased 413 folds. Till now this is the highest purification yield obtained for any purified pediocin. During purification, it was shown that the peptide was having enhanced antimicrobial activity at acidic pH than at neutral pH. Both examination of the inhibitory spectrum and the characterization of AMPs do not give sufficient and exact information to distinguish between different pediocins. Therefore, it may be recommended to include PCR based screening as a preliminary step to detect new pediocins or more accurately AMPs. In Silico analysis showed that pediocin 273 is a positively charged, hydrophobic, YGNGV motif containing antimicrobial peptide. Pediocin 273 suggests a great potential in applications in food industry, pharmaceutics and biomedicine. [PUBLICATION ABSTRACT]

BackgroundTo adopt HPV self-sampling in Japan, we assessed the concordance between self- and physician-collected human papillomavirus (HPV) samples from Japanese patients and examined the performance of HPV self-sampling for cervical intraepithelial neoplasia grade 2 or worse (CIN2+).MethodsPatients who had previously tested negative for intraepithelial lesions or malignancy/HPV-positive, and patients with atypical squamous cells of undetermined significance or worse (ASCUS+) cytology were eligible for this cross-sectional study. Participants performed HPV self-sampling using an Evalyn brush, which was submitted at the Fukui Prefectural Health Care Association. The Evalyn brush heads were stored in ThinPrep vials. The physician, however, performed HPV and cell sampling using an endocervical brush and immediately stored the brush heads in ThinPrep vials. All participants underwent colposcopy and biopsy. Histopathological diagnoses were made by pathologists at Fukui University Hospital. HPV infection was confirmed using a PCR-based Cobas 4800 HPV DNA test. Cytological analysis was performed at Fukui Prefectural Health Care Association.ResultsHPV-positive rates for physician-collected samples and self-collected samples were 51 and 50%, respectively. The perfect match rate of HPV type between the groups was 88% (κ = 0.76). HPV16/18 showed higher agreement rates than other HPVs (99%, kappa 0.96 and 89% kappa 0.77, respectively). Both groups showed 100% sensitivity to CIN2+, but specificity was 57.0 and 58.1%, respectively.ConclusionFor HPV typing, a good concordance rate was seen between self- and physician-collected samples. Self-sampling showed high sensitivity for CIN2+. Self-sampling using the Evalyn brush and Cobas 4800 may be feasible for screening Japanese individuals.

Phosphorus (P) is the second most crucial nutrient for plant growth after nitrogen. However, its highly reactive nature causes formation of insoluble derivatives and limits uptake by the plant roots. The wide spread applications of P based chemical fertilizers cause detrimental effects on soil fertility, agricultural product quality and environment. In this regard, phosphate-solubilizing microorganisms (PSMs) stand out as the most remarkable and promising tools for the development of safer and sustainable technologies. As a result of this, many bacterial and fungal species with significant phosphate-solubilizing activity have been discovered by using the conventional screening methods. However, the growing need for the discovery of new strains of PSMs necessitates the replacement or support to the time-consuming conventional methods with techniques that are more sensitive, reliable, reproducible and less time consuming. In this context, molecular tools and techniques provide novel approaches for microbial phosphate solubilization research. Hence, in this review information on the molecular approaches for the PSMs research is provided and its importance explained. The review also discusses the genes related to phosphate solubilizing mechanisms and molecular tools for screening these genes.

Cervical cancer incidence is increasing among Japanese women, which is partly attributed to low screening rates. This study examined the implementation of opt-in human papillomavirus (HPV) self-sampling among Japanese women aged 30–39 years who had not undergone cervical cancer screening, focusing on those requiring preconception care. The responses to the opt-in approach and effectiveness in detecting cervical squamous intraepithelial neoplasia 2 or worse (CIN2+) were evaluated. Participants used the Evalyn® Brush for self-sampling, with HPV testing conducted using the Cobas 4800 system (version 2.2.0). Out of 3489 eligible, unscreened women from four municipalities in Fukui Prefecture, only 10.6% (370/3489) requested the self-sampling kit. Of these, 77.3% (286/370) returned the kit (HPV testing rate: 8.2% (286/3489)). The HPV positivity rate was 13.7% (39/285), yet only 61.5% (24/39) of those with positive HPV results proceeded to cytology testing. Subsequently, three cases of CIN2+ were detected (10.5/1000). While this study demonstrated a reasonable kit return rate and indicated the capability of opt-in HPV self-sampling to detect CIN2+ cases in unscreened women, the low ordering rate of kits and suboptimal compliance for follow-up cytology testing highlight significant challenges. The findings suggest the need for more effective strategies to enhance participation in cervical cancer screening programs.

Background: The increasing trend of cervical cancer in women in their 20s in Japan is largely attributable to the low rate of cervical cancer screening. This study aimed to assess the usefulness of human papillomavirus (HPV) self-sampling among 24-year-old Japanese women who had never previously been screened for cervical cancer during the coronavirus disease (COVID-19) pandemic. Methods: In August 2021, consenting eligible women received HPV self-sampling kits. An Evalyn brush was used for self-sampling, and a Cobas 4800 PCR-based HPV DNA test was used to detect high-risk HPV genotypes. We analyzed the return rates of self-sampling kits and conducted a survey on the acceptability of the self-sampling method. Results: Of the total 1997 eligible women, 13.4% (268/1997) agreed to participate. The return rate of the kits was 72.4% (194/268), corresponding to 9.7% of the eligible population. Among the participants who returned the kits, 14.9% (29/194) tested positive for HPV, and 41.4% (12/29) of these underwent subsequent cytological testing. The questionnaire results indicated that 57.8% of participants reported no pain during self-sampling, and 72.9% expressed a willingness to continue using the self-sampling method in the future. Conclusion: This study demonstrated that opt-in HPV self-sampling among 24-year-old women who had never been screened for cervical cancer had a favorable kit return rate and was well accepted by the participants, especially during the COVID-19 pandemic. However, the follow-up cytology test rates were low, highlighting the need for improved post-screening management.

Advances in genetic technologies have made genetic testing more accessible than ever before. However, depending on national, regional, legal, and health insurance circumstances, testing procedures may still need to be streamlined in real-world clinical practice. In cases of autosomal recessive disease with consanguinity, the mutation locus is necessarily isodisomy because both alleles originate from a common ancestral chromosome. Based on this premise, we implemented integrated genetic diagnostic methods using SNP array screening and long range PCR-based targeted NGS in a Japanese patient with xeroderma pigmentosum (XP) under the limitation of the national health insurance system. SNP array results showed isodisomy only in XPC and ERCC4 loci. NGS, with a minimal set of long-range PCR primers, detected a homozygous frameshift mutation in XPC; NM_004628.5:c.218_219insT p.(Lys73AsnfsTer9), confirmed by Sanger sequencing, leading to a rapid diagnosis of XP group C. This shortcut strategy is applicable to all autosomal recessive diseases caused by consanguineous marriages, especially in scenarios with a moderate number of genes to test, a common occurrence in clinical genetic practice.

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum , and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9 , cf-9 , Cf-0 , and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9 , Cf-0 , and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum .

Stink bugs (Hemiptera: Pentatomidae) are economic pests of cotton and soybean. This study was conducted to examine predation on stink bugs by arthropod predators in cotton and adjoining soybean habitats. Gut-content analysis based on polymerase chain reaction (PCR) was used to detect stink bug deoxyribonucleic acid (DNA) in predators collected from both crops over a 5 wk period. Nezara viridula (L.), Euschistus servus (Say), Chinavia hilaris (Say), and Euschistus quadrator Rolston were detected on soybean and cotton. Piezodorus guildinii (Westwood) and Thyanta custator custator (F.) were detected only on soybean whereas Euschistus tristigmus (Say) was detected only on cotton. Over both crops, 13 predators screened positive for a variety of stink bug species DNA by PCR analysis: Geocoris punctipes (Say) and Geocoris uliginosus (Say) (Hemiptera: Geocoridae), Orius insidiosus (Say) (Hemiptera: Anthocoridae), Hippodamia convergens Guérin-Méneville, Harmonia axyridis (Pallas) (cotton), and Scymnus sp. (cotton) (Coleoptera: Coccinellidae), Oxyopes salticus Hentz and Peucetia viridans (Hentz) (cotton) (Araneae: Oxyopidae), Solenopsis invicta Buren (Hymenoptera: Formicidae), Podisus maculiventris (Say) (Hemiptera: Pentatomidae), Mecaphesa asperata (Hentz) (Araneae: Thomisidae), Zelus renardii Kolenati (Hemiptera: Reduviidae), and Notoxus monodon (F.) (cotton) (Coleoptera: Anthicidae). In soybean, the percentage of G. punctipes and G. uliginosus screening positive for N. viridula was high, 87.3%, whereas the percentage screening positive for E. servus was moderately high, 60.3%. In cotton, the percentage of N. viridula DNA in gut-contents of O. insidiosus was high, 91.6%. Detection of P. guildinii and/or T. c. custator DNA in predators in cotton and of E. tristigmus DNA in predators in soybean demonstrated predator dispersal between soybean and cotton. In soybean, the percentage of P. guildinii DNA in gut contents of G. punctipes, G. uliginosus, and O. insidiosus, including those individuals in cotton that dispersed from soybean, was high. We conclude that a complex of arthropod predators prey on a complex of stink bugs in both cotton and adjoining soybean while foraging in and between these crops.