Mutant firefly luciferases with improved specific activity and dATP discrimination constructed by yeast cell surface engineering

Pyrosequencing system utilizing luciferase is one of the next-generation DNA sequencing systems. However, there is a crucial problem with the current pyrosequencing system: luciferase cannot discriminate between ATP and dATP completely, and dATPαS must be used as the dATP analogue. dATPαS is expensive and has low activity for the enzyme. If luciferase can clearly recognize the difference between ATP and dATP, dATP could be used instead of the expensive dATPαS in the pyrosequencing system. We attempted to prepare a novel luciferase with improved specific activity and dATP discrimination with the molecular display method. First, we selected two amino acid residues, Ser440 and Ser456, as target residues for mutation from the whole sequence of Photinus pyralis luciferase; we comprehensively mutated these two amino acids. A mutant luciferase library was constructed using yeast cell surface engineering. Through three step-wide screenings with individual conditions, we easily and speedily isolated three candidate mutants from 1,152 candidates and analyzed the properties of these mutants. Consequently, we succeeded in obtaining interesting mutant luciferases with improved specific activity and dATP discrimination more conveniently than with other methods.     

Uniparental disomy analysis in trios using genome-wide SNP array and whole-genome sequencing data imply segmental uniparental isodisomy in general populations

Whole chromosomal and segmental uniparental disomy (UPD) is one of the causes of imprinting disorder and other recessive disorders. Most investigations of UPD were performed only using cases with relevant phenotypic features and included few markers. However, the diagnosis of cases with segmental UPD requires a large number of molecular investigations. Currently, the accurate frequency of whole chromosomal and segmental UPD in a normal developing embryo is not well understood. Here, we present whole chromosome and segmental UPD analysis using single nucleotide polymorphism (SNP) microarray data of 173 mother-father-child trios (519 individuals) from six populations (including 170 HapMap trios). For two of these trios, we also investigated the possibility of shorter segmental UPD as a consequence of homologous recombination repair (HR) for DNA double strand breaks (DSBs) during the early developing stage using high-coverage whole-genome sequencing (WGS) data from 1000 Genomes Project. This could be overlooked by SNP microarray. We identified one obvious segmental paternal uniparental isodisomy (iUPD) (8.2 mega bases) in one HapMap sample from 173 trios using Genome-Wide Human SNP Array 6.0 (SNP6.0 array) data. However, we could not identify shorter segmental iUPD in two trios using WGS data. Finally, we estimated the rate of segmental UPD to be one per 173 births (0.578%) based on the UPD screening for 173 trios in general populations. Based on the autosomal chromosome pairs investigated, we estimate the rate of segmental UPD to be one per 3806 chromosome pairs (0.026%). These data imply the possibility of hidden segmental UPD in normal individuals.     

The association between circadian rest-activity patterns and the behavioral and psychological symptoms depending on the cognitive status in Japanese nursing-home residents

ABSTRACT

Background: Limited information is available on the relationship between sleep disturbances during nighttime and the behavioral and psychological symptoms of dementia in older nursing-home residents. However, a few reports on the association between the circadian rest-activity rhythm and the behavioral and psychological symptoms of dementia in older residents have been published. The main objective of the present study was to examine the association among the circadian rest-activity rhythm, behavioral and psychological symptoms, and the cognitive function status among older individuals living in facilities. Method: The investigation was conducted from September 2017 to February 2018, and participants were recruited from five nursing homes in Akita prefecture, Japan, after obtaining patient agreement to participate in the study. To measure nonparametric circadian rest-activity parameters such as interdaily stability, intradaily variability, relative amplitude, mean of the least active 5-h period, and mean of the most active 10-h period, Actigraph devices were worn on the participants’ nondominant wrists continuously for seven days. The score or classification of the cognitive status and the severity of the behavioral and psychological symptoms of dementia (BPSD) were assessed using the clinical dementia rating (CDR) and the dementia behavior disturbance scale (DBD), respectively. The binomial logistic regression model was applied to clarify which kinds of circadian rest-activity parameters predicted the cognitive status in nursing home residents as well as the BPSD outcome. A multi-level model was also used to examine the association between the nonparametric rest-activity parameters and the BPSD outcome explained by the cognitive status among older individuals in facilities. Results: Seventy-seven participants (49 residents with dementia, and 28 residents without dementia) were included in this study. According to the binomial logistic regression analysis after adjusting for covariates, the classification of the cognitive status for older residents was associated with the DBD score (odds ratio, 1.22; 95% confidence interval [CI], 1.08, 1.38; p < 0.001), the IS (odds ratio, 0.01; 95% CI, 0.00, 1.00; p = 0.05) and the L5 (odds ratio, 0.99; 95% CI, 0.99, 1.00; p = 0.05). The results of a multi-level model also indicated that the IV at individual-level was significantly associated with the DBD score for nursing home residents, with the CDR score at cluster-level as an explanatory variable. As well, a significant association between the RA at individual level and the DBD score was observed in a multi-level model explained by the CDR score at cluster-level. Conclusion: Of these models, the multi-level model provided grounds for our proposal that the fragmentation or the amplitude of rest-activity parameters might be associated with the outcome of BPSD, considering the cognitive status of older individuals in different facilities. The findings offer practical insight into the prevention of BPSD and the improvement of rest-activity rhythms in rehabilitative care in nursing homes.     

Gastrointestinal passage time of seeds ingested by captive Japanese martens <Emphasis Type="Italic">Martes melampus</Emphasis>

The time it takes for ingested seeds to pass through the gut of animals is an important aspect of endozoochorous seed dispersal because it influences seed dispersal distance. Variations in the physical characteristics of seeds, such as their weight, volume, and specific gravity, can affect their movement through the gastrointestinal system of a given animal. We conducted feeding experiments with captive Japanese martens, Martes melampus (n = 4), at Toyama Municipal Family Park Zoo, central Japan to examine the effects of the physical characteristics of seeds on their passage times. The mean (±SD) transit time, mean retention time, and time of last appearance of four different types of commercial seeds were 2.6 ± 0.3 h (range 0.6–5.4), 9.7 ± 1.1 h (3.8–17.3), and 23.8 ± 3.1 h (12.2–51.8), respectively. All of these values are greater than those found during previous experiments conducted with mustelids. Similar to previous studies, however, none of these passage time variables was correlated with the physical characteristics of seeds. Our results thus indicate that martens disperse seeds of different plant species, whose size, volume, and specific gravity all fall within the range of those used in the present study, from parent plants at similar distances.     

TRPV1 agonist piperine but not olvanil enhances glutamatergic spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons

We examined the effects of TRPV1 agonists olvanil and piperine on glutamatergic spontaneous excitatory transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices with the whole-cell patch-clamp technique. Bath-applied olvanil did not affect the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC), and unchanged holding currents at −70 mV. On the other hand, superfusing piperine reversibly and concentration-dependently increased sEPSC frequency (half-maximal effective concentration: 52.3 μM) with a minimal increase in its amplitude. This sEPSC frequency increase was almost repetitive at an interval of more than 20 min. Piperine at a high concentration produced an inward current in some neurons. The facilitatory effect of piperine was blocked by TRPV1 antagonist capsazepine. It is concluded that piperine but not olvanil activates TRPV1 channels in the central terminals of primary-afferent neurons, resulting in an increase in the spontaneous release of l-glutamate onto SG neurons.     

Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo

Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences.The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations.In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal kinase 1/2 (p-JNK1/2) were strongly observed in the marginal layer and the dorsal horn from E13 to E17. Our results suggest that p-ERK1/2 and p-JNK1/2 play critical roles in the developing spinal cord. Constructing phosphoprotein atlases will be possible in the future if this work is systematically developed on a larger scale than we presented here.     

Previous Helicobacter pylori infection–induced atrophic gastritis: A distinct disease entity in an understudied population without a history of eradication

Individuals with chronic atrophic gastritis who are negative for active H. pylori infection with no history of eradication therapy have been identified in clinical practice. By excluding false‐negative and autoimmune gastritis cases, it can be surmised that most of these patients have experienced unintentional eradication of H. pylori after antibiotic treatment for other infectious disease, unreported successful eradication, or H. pylori that spontaneously disappeared. These patients are considered to have previous H. pylori infection–induced atrophic gastritis. In this work, we define these cases based on the following criteria: absence of previous H. pylori eradication; atrophic changes on endoscopy or histologic confirmation of glandular atrophy; negative for a current H. pylori infection diagnosed in the absence of proton‐pump inhibitors or antibiotics; and absence of localized corpus atrophy, positivity for autoantibodies, or characteristic histologic findings suggestive of autoimmune gastritis. The risk of developing gastric cancer depends on the atrophic grade. The reported rate of developing gastric cancer is 0.31%‐0.62% per year for successfully eradicated severely atrophic cases (pathophysiologically equal to unintentionally eradicated cases and unreported eradicated cases), and 0.53%‐0.87% per year for spontaneously resolved cases due to severe atrophy. Therefore, for previous H. pylori infection–induced atrophic gastritis cases, we recommend endoscopic surveillance every 3 years for high‐risk patients, including those with endoscopically severe atrophy or intestinal metaplasia. Because of the difficulty involved in the endoscopic diagnosis of gastric cancer in cases of previous infection, appropriate monitoring of the high‐risk subgroup of this understudied population is especially important.