Expanding the Repertoire of Gene Tools for Precise Manipulation of the Clostridium difficile Genome: Allelic Exchange Using pyrE Alleles

Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the ‘correction’ method (5–7 days) makes pyrE ⁻ strains attractive hosts for mutagenesis studies.     

A novel synthesis and antimicrobial activity of 1-[(Substituted-phenyl) sulfonyl]pyrrolidin-2-ones

Novel cyclization of 4-(substituted-phenylsulfonamido)butanoic acids to their corresponding 1-[(substituted-phenyl)sulfonyl]pyrrolidin-2-ones was successfully achieved by using polyphosphate ester (PPE). The reaction time was considerably reduced with corresponding increase in the yields, when polyphosphate ester (PPE) was used in combination with 4-(N,N-dimethylamino)pyridine (DMAP). All the synthesized compounds were screened for their antimicrobial activity. Minimum Inhibitory Concentration (MIC) values of synthesized compounds were also determined, and were found to be in the range of 0.09-1.0 mg.     

Design,synthesis and anti-melanogenic effect of cinnamamide derivatives

Pigmentation disorders are attributed to excessive melanin which can be produced by tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders associated with overpigmentation. Based on our previous findings that an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and 10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl substituent showed much higher mushroom tyrosinase inhibition at 25?µM than kojic acid (18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9 having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10 murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was a close correlation between cellular tyrosinase activity and melanin content, indicating that the inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the (E)-β-phenyl-α,β-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents.     

<Emphasis Type="Italic">Brevibacterium anseongense</Emphasis> sp. nov., isolated from soil of ginseng field

Gram-positive, aerobic, non-motile, pale-yellow, and rodshaped bacterium, designated as Gsoil 188^T, was isolated from the soil of a ginseng field in Pocheon, South Korea. A phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Brevibacterium and was most closely related to B. epidermidis NBRC 14811^T (98.4%), B. sediminis FXJ8.269^T (98.2%), B. avium NCFB 3055^T (98.1%), and B. oceani BBH7^T (98.1%), while it shared less than 98.1% identity with the other species of this genus. The DNA G + C content was 68.1 mol%. The predominant quinone was MK-8(H₂). The major fatty acids were anteiso-C_15:0 and anteiso-C_17:0. The cell wall peptidoglycan of strain Gsoil 188^T contained meso-diaminopimelic acid. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminolipid. The physiological and biochemical characteristics, low DNA-DNA relatedness values, and taxonomic analysis allowed the differentiation of strain Gsoil 188^T from the other recognized species of the genus Brevibacterium. Therefore, strain Gsoil 188^T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium anseongense sp. nov. is proposed, with the type strain Gsoil 188^T (= KACC 19439^T = LMG 30331^T).     

Study of rust resistance genes in wheat germplasm with DNA markers

Wheat is a vital dietary component for human health and widely consumed in the world. Wheat rusts are dangerous pathogens and contribute serious threat to its production. In present study, PCR-Based DNA Markers were employed to check the rust resistance genes among 20 wheat genotypes and 22 markers were amplified. NTSYS-pc 2.2 was used to calculate genetic diversity and Nei and Li''s coefficients ranged from 0.55 to 0.95. Cluster analysis was obtained using UPGMA (Unweighted Pair Group Method of Arithmetic Average) algorithm. Maximum no. of genes (23) was amplified from TW-760010 genotype whereas minimum no of genes (14) were amplified from TW-76005 genotype. The data gained from present study open up new ways to produce new varieties by breeding rust resistant germplasm to avoid the economic and food loss and varieties with improved characteristics.     

P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics

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Anaplastic thyroid cancer: outcomes of trimodal therapy

BackroundThe purpose of this study is to assess the impact of trimodal therapy [surgery, chemotherapy and external beam radiotherapy (EBRT)] in patients with anaplastic thyroid cancer (ATC) treated with curative intent.Materials and methodsRetrospective review of patients with ATC treated at a tertiary referral centre between January 2009 and June 2020. Data were collected regarding demographics, histology, staging, treatment and outcomes.ResultsSeven patients (4 female) were identified. Median age was 58 years (range 52–83 years). All patients received EBRT with concurrent doxorubicin. Six patients received surgery followed by chemoradiotherapy (CRT), and one underwent neoadjuvant CRT followed by surgery. Median radiological tumour size was 50mm (range 40–90 mm). Six patients had gross extrathyroidal extension and three had N1b disease. Prescribed radiotherapy schedules were 46.4 Gy in 29 bidaily fractions (n = 2, treated 2010), 60 Gy in 30 daily fractions (n = 2), 66 Gy in 30 fractions (n = 2) and 70 Gy in 35 fractions (n = 1; patient received neoadjuvant CRT). CRT was discontinued early for two patients due to toxicities. At median follow up of 5.8 months, 42.9% (3/7) patients were alive and disease-free. Only one patient developed a local failure. Three patients died from distant metastases without locoregional recurrence.ConclusionsDespite poor prognosis of ATC, selected patients with operable tumours may achieve high locoregional control rates with trimodal therapy, with possibility of long-term survival in select cases.     

Synthesis and fungicidal activity of novel pyrazole derivatives containing 5-Phenyl-2-Furan

Pyrazole constitutes an important heterocyclic family covering a broad range of synthetic as well as natural products that exhibit numerous chemical, biological, agrochemical and pharmacological properties. In order to explore compounds with good fungicidal activity, a series of new pyrazole derivatives containing 5-phenyl-2-furan were designed and synthesized. In vitro and in vivo fungicidal activities were evaluated and the compound ethyl-1-(5-phenylfuran-2-carbonyl)-5-propyl-1H-pyrazole-3-carboxylate (I8) displayed significant fungicidal activity against various fungi, especially against P. infestans. The structures of the novel pyrazole derivatives were confirmed by ¹H NMR, ¹³C NMR, MS, elemental analysis and X-ray single crystal diffraction. Further study showed that compound I8 might act on the synthesis of cell walls from morphological and ultrastructural studies by SEM and TEM. The results also revealed that compound I8 could block the nutritional transportation leading to cells senescence and death. These results suggested that the novel pyrazole derivatives proved to be promising lead compounds.