Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase δ

A novel mutagenesis technique using error-prone DNA polymerase δ (polδ), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the polδMKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant polδ supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the polδMKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis

Denoising using deep-learning-based reconstruction for whole-heart coronary MRA with sub-millimeter isotropic resolution at 3 T: a volunteer study

PURPOSEThe aim of this study was to assess the usefulness of denoising deep-learning-based reconstruction (dDLR) to improve image quality and vessel delineation in noncontrast 3-T wholeheart coronary magnetic resonance angiography (WHCMRA) with sub-millimeter isotropic resolution (Sub-mm) compared with a standard resolution without dDLR (Standard).METHODSFor 10 healthy volunteers, we acquired the WHCMRA with Sub-mm with and without dDLR and Standard to quantify signal- (SNR) and contrast-to-noise ratio (CNR) and vessel edge signal response (VESR) in all the 3 image types. Two independent readers subjectively graded vessel sharpness and signal homogeneity of 8 coronary segments in each patient. We used Kruskal– Wallis test with Bonferroni correction to compare SNR, CNR, VESR, and the subjective evaluation scores among the 3 image types and weighted kappa test to evaluate inter-reader agreement on the scores.RESULTSSNR was significantly higher with Sub-mm with dDLR (P .05); the subjective signal homogeneity was significantly improved from Sub-mm without dDLR to Standard to Sub-mm with dDLR (P < .001). The inter-reader agreement was excellent (kappa=0.84).CONCLUSIONApplication of dDLR is useful for improving image quality and vessel delineation in the WHCMRA with Sub-mm compared with Standard

A non-carboxylating pentose bisphosphate pathway in halophilic archaea

Bacteria and Eucarya utilize the non-oxidative pentose phosphate pathway to direct the ribose moieties of nucleosides to central carbon metabolism. Many archaea do not possess this pathway, and instead, Thermococcales utilize a pentose bisphosphate pathway involving ribose-1, 5-bisphosphate (R15P) isomerase and ribulose-1, 5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Intriguingly, multiple genomes from halophilic archaea seem only to harbor R15P isomerase, and do not harbor Rubisco. In this study, we identify a previously unrecognized nucleoside degradation pathway in halophilic archaea, composed of guanosine phosphorylase, ATP-dependent ribose-1-phosphate kinase, R15P isomerase, RuBP phosphatase, ribulose-1-phosphate aldolase, and glycolaldehyde reductase. The pathway converts the ribose moiety of guanosine to dihydroxyacetone phosphate and ethylene glycol. Although the metabolic route from guanosine to RuBP via R15P is similar to that of the pentose bisphosphate pathway in Thermococcales, the downstream route does not utilize Rubisco and is unique to halophilic archaea

Reassessment of the In Vivo Functions of DNA Polymerase I and RNase H in Bacterial Cell Growth▿ †

A major factor in removing RNA primers during the processing of Okazaki fragments is DNA polymerase I (Pol I). Pol I is thought to remove the RNA primers and to fill the resulting gaps simultaneously. RNase H, encoded by rnh genes, is another factor in removing the RNA primers, and there is disagreement with respect to the essentiality of both the polA and rnh genes. In a previous study, we looked for the synthetic lethality of paralogs in Bacillus subtilis and detected several essential doublet paralogs, including the polA ypcP pair. YpcP consists of only the 5′-3′ exonuclease domain. In the current study, we first confirmed that the polA genes of both Escherichia coli and B. subtilis could be completely deleted. We found that the 5′-3′ exonuclease activity encoded by either polA or ypcP xni was required for the growth of B. subtilis and E. coli. Also, the 5′-3′ exonuclease activity of Pol I was indispensable in the cyanobacterium Synechococcus elongatus. These results suggest that a 5′-3′ exonuclease activity is essential in these organisms. Our success in constructing a B. subtilis strain that lacked all RNase H genes indicates that the enzymatic activity is dispensable, at least in the wild type. Increasing the 5′-3′ exonuclease activity partially compensated for a defective phenotype of an RNase H-deficient mutant, suggesting cooperative functions for the two enzyme systems. Our search for the distribution of the 5′-3′ exonuclease domain among 250 bacterial genomes resulted in the finding that all eubacteria, but not archaea, possess this domain

Myoepithelial carcinoma of the parotid gland: A case of adequate fine-needle aspiration cytology specimens rendering a conclusive diagnosis possible

An 80-year-old male presented with a history of a hard right parotid mass that had gradually increased in size, with subsequent facial paralysis. A fine-needle aspiration biopsy was performed. The cytologic specimens contained a substantial number of sheet-like clusters or small groups of a mixture of plasmacytoid, oval to spindled, or large epithelioid cells having hyperchromatic pleomorphic nuclei, abundant cytoplasm with occasional inclusion body-like materials, and prominent nucleoli, in a relatively clear background. We first interpreted it as a carcinoma, suggestive of myoepithelial differentiation. Radical parotidectomy was performed, and a gross examination of the neoplasm revealed a non-capsulated and ill-defined tumor lesion, with a grayish or yellowish cut surface, associated with fat invasion. On a microscopic examination, the tumor was predominantly composed of the solid proliferation of atypical cells including a mixture of oval to spindled, plasmacytoid, or epithelioid cells, often arranged in a trabecular and reticular growth pattern with patchy eosinophilic hyalinized stroma. Immunohistochemistry showed that the carcinoma cells were specifically positive for p63, cytokeratins, and vimentin. Finally, electron microscopy demonstrated that their phenotype was consistent with a myoepithelial origin containing many bundles of variably thin actin filaments. Therefore, we finally made a diagnosis of myoepithelial carcinoma, defined as the malignant counterpart of benign myoepithelioma. We should be aware that owing to its characteristic cytological features, cytopathologists may be able to make a correct diagnosis of myoepithelial carcinoma, based on multiple and adequate samplings

Analysis of the Anticipatory Behavior Formation Mechanism Induced by Methamphetamine Using a Single Hair

While the suprachiasmatic nucleus (SCN) coordinates many daily rhythms, some circadian patterns of expression are controlled by SCN-independent systems. These include responses to daily methamphetamine (MAP) injections. Scheduled daily injections of MAP resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. The MAP-induced anticipatory behavior is associated with the induction and a phase advance in the expression rhythm of the clock gene Period1 (Per1). However, this unique formation mechanism of MAP-induced anticipatory behavior is not well understood. We recently developed a micro-photomultiplier tube (micro-PMT) system to detect a small amount of Per1 expression. In the present study, we used this system to measure the formation kinetics of MAP-induced anticipatory activity in a single whisker hair to reveal the underlying mechanism. Our results suggest that whisker hairs respond to daily MAP administration, and that Per1 expression is affected. We also found that elevated Per1 expression in a single whisker hair is associated with the occurrence of anticipatory behavior rhythm. The present results suggest that elevated Per1 expression in hairs might be a marker of anticipatory behavior formation