New method for comparing DNA primary sequences based on a discrimination measure

We introduce a new approach to compare DNA primary sequences. The core of our method is a new measure of pairwise distances among sequences. Using the primitive discrimination substrings of sequence S and Q, a discrimination measure DM(S, Q) is defined for the similarity analysis of them. The proposed method does not require multiple alignments and is fully automatic. To illustrate its utility, we construct phylogenetic trees on two independent data sets. The results indicate that the method is efficient and powerful.     

IPCA-CMI: An Algorithm for Inferring Gene Regulatory Networks based on a Combination of PCA-CMI and MIT Score

Inferring gene regulatory networks (GRNs) is a major issue in systems biology, which explicitly characterizes regulatory processes in the cell. The Path Consistency Algorithm based on Conditional Mutual Information (PCA-CMI) is a well-known method in this field. In this study, we introduce a new algorithm (IPCA-CMI) and apply it to a number of gene expression data sets in order to evaluate the accuracy of the algorithm to infer GRNs. The IPCA-CMI can be categorized as a hybrid method, using the PCA-CMI and Hill-Climbing algorithm (based on MIT score). The conditional dependence between variables is determined by the conditional mutual information test which can take into account both linear and nonlinear genes relations. IPCA-CMI uses a score and search method and defines a selected set of variables which is adjacent to one of or Y. This set is used to determine the dependency between X and Y. This method is compared with the method of evaluating dependency by PCA-CMI in which the set of variables adjacent to both X and Y, is selected. The merits of the IPCA-CMI are evaluated by applying this algorithm to the DREAM3 Challenge data sets with n variables and n samples () and to experimental data from Escherichia coil containing 9 variables and 9 samples. Results indicate that applying the IPCA-CMI improves the precision of learning the structure of the GRNs in comparison with that of the PCA-CMI.     

In vivo cloning of DNA regions carrying mutations linked to selectable genes: application to mutations in the regulatory region of the Escherichia coli tryptophan operon.

A rapid method for cloning DNA regions carrying specific mutations onto a previously characterized plasmid is described. This method is used to clone DNA regions carrying trpL or trpP mutations in the Escherichia coli tryptophan operon. The applicability of the method for other systems is discussed.     

Estimation of coalescence probabilities and population divergence times from SNP data

We present a method called the G(A|B) method for estimating coalescence probabilities within population lineages from genome sequences when one individual is sampled from each population. Population divergence times can be estimated from these coalescence probabilities if additional assumptions about the history of population sizes are made. Our method is based on a method presented by Rasmussen et al. (2014) to test whether an archaic genome is from a population directly ancestral to a present-day population. The G(A|B) method does not require distinguishing ancestral from derived alleles or assumptions about demographic history before population divergence. We discuss the relationship of our method to two similar methods, one introduced by Green et al. (2010) and called the F(A|B) method and the other introduced by Schlebusch et al. (2017) and called the TT method. When our method is applied to individuals from three or more populations, it provides a test of whether the population history is treelike because coalescence probabilities are additive on a tree. We illustrate the use of our method by applying it to three high-coverage archaic genomes, two Neanderthals (Vindija and Altai) and a Denisovan.Subject terms: Rare variants, Evolutionary genetics

One of the goals of population genetics is to estimate the divergence time of isolated populations. We will review several methods that have been proposed and present a new method that is closely related to two existing methods. We will emphasize the assumptions made when using different methods. It will be useful to make the distinction between estimating coalescence probabilities within populations and estimating population divergence times. We will also introduce a test for a treelike population history based on our method.For distantly related populations, the numbers of mutational differences between sequences indicate relative times of divergence. Relative times are converted to absolute times by assuming a mutation rate. This method traces to Zuckerkandl and Pauling (1962, 1965) and has been used and refined extensively. This class of methods estimates genomic divergence times. Using it to estimate population or species divergence times assumes that those times are so large that the difference between them can be ignored.For recently diverged populations, the numbers of mutational differences probably do not provide a reliable estimate of population divergence times both because there may be too few mutations that differentiate populations and because the difference between the genomic and population divergence times may be substantial. To overcome this problem, Green et al. (2010) (in Supplement 14) introduced a method that accounts for the difference between genomic and population divergence. This method was used in later papers from the same group (Meyer et al. 2012; Prüfer et al. 2014, 2017).The Green et al. (2010) method is applicable when one genome is sampled from each of two populations. It depends on the statistic F(A|B), which is the fraction of sites in population A that carry the derived allele when that site is heterozygous in population B. Green et al. (2010) showed by simulation that the expectation of F(A|B) decreases roughly exponentially with the separation time of A and B. The rate of decrease depends on the history of population sizes both in B and in the population ancestral to A and B. Green et al. (2010) estimated population divergence times by interpolating their simulation results.More recently, Schlebusch et al. (2017), in Section 9.1 of their supplementary materials, introduced a similar method, called the TT method. Their method is based on analytic expressions for the configuration probabilities of SNPs that are polymorphic in the two populations. The TT method assumes that ancestral and derived alleles can be distinguished and the population before divergence was of constant size. The TT method is developed and elaborated on by Sjödin et al. (2020).In the present paper, we present a new method that is closely related to the F(A|B) and TT methods. We call it the G(A|B) method to emphasize its similarity to F(A|B). Our method is based on a method presented by Rasmussen et al. (2014) to test whether an ancient DNA sequence is from a population directly ancestral to a present-day population. We will show that our method provides a way to test whether the history of three or more populations is accurately represented by a population tree even if the demographic histories of those populations are not known.     

Ultra-performance liquid chromatography fingerprint combined with chemometrics as an effective strategy for Dianbaizhu species discrimination

Dianbaizhu a folk medicine used for the treatment of rheumatoid arthritis is made from different species of Gaultheria. The main species is Gaultheria leucocarpa var. yunnanensis but other species are being used as substitutes. The objective was to develop a precise and reproducible method to differentiate among these species. A method was developed by using ultra-performance liquid chromatography in combination with chemometric. The method was validated with relative standard deviations of less than 1.96%.     

Novel method for determination of phenol degradation kinetics

In this study, we proposed a new method for estimating biokinetic parameters in phenol degradation kinetics. The new method relies on the new formulation of q–S relation where degradation rate q is calculated from the changes of substrate concentration S for each time segment during the course of entire degradation, while in the conventional method q is obtained from the slope of the straight line that is given as substrate concentration changes with time in a semi-logarithmic scale. Thus, this new method provided more data points than the conventional method. The q–S relations obtained from the new method and the conventional method were fitted with three inhibitory kinetic models of Haldane, Yano and Edwards. Simulation of degradation profile with each kinetic model and comparison with the observed profile revealed that the new method offered a better prediction with Edwards model as the best inhibitory model.     

Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

Bio-diesel production directly from the microalgae biomass of Nannochloropsis by microwave and ultrasound radiation

This work offers an optimized method for the direct conversion of harvested Nannochloropsis algae into bio-diesel using two novel techniques. The first is a unique bio-technology-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation. This method reduces considerably the cost of algae production. The second technique is the direct transesterification (a one-stage method) of the Nannochloropsis biomass to bio-diesel production using microwave and ultrasound radiation with the aid of a SrO catalyst. These two techniques were tested and compared to identify the most effective bio-diesel production method. Based on our results, it is concluded that the microwave oven method appears to be the most simple and efficient method for the one-stage direct transesterification of the as-harvested Nannochloropsis algae.     

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer

The increasing Yezo sika deer (Cervus nippon yesoensis) population is creating a large problem. Yezo sika deer are an important blood meal source, and these deer contribute to the maintenance of tick populations. Theileria spp. infections in Yezo sika deer and T. orientalis infections in cows occur at high frequencies, and the same tick species infests both deer and cows. Therefore, a specific detection method to identify deer Theileria spp. is important. In this study, we establish a novel molecular detection method for identifying Theileria spp. from deer and tick samples using loop-mediated isothermal amplification (LAMP). This method targets a metalloprotease/cell division cycle protein gene homologue. Our LAMP protocol was able to detect deer Theileria and did not show cross reactivity with other closely related protozoan parasites, including T. orientalis. The LAMP method showed sensitivity and specificity equivalent to those of nested PCR performed on the same field samples from deer and ticks. These results demonstrate the applicability of LAMP to field surveys in which the detection of deer Theileria spp. is required. In conclusion, due to its simplicity, specificity, and reliability, we suggest our LAMP protocol as an appropriate method for routine surveys to detect Yezo sika deer and ticks infected with deer Theileria spp. parasites. Additionally, this LAMP method offers great promise as a useful tool to distinguish Yezo sika deer Theileria from related Theileria parasites present in livestock.     

A simplified diphenylamine colorimetric method for growth quantification

Cell growth needs to be monitored in biological studies and bioprocess optimization. In special circumstances, such as microbial fermentations in media containing insoluble particles, accurate cell growth quantification is a challenge with current methods. Only the Burton method is applicable in such circumstances. The original Burton method was previously simplified by adopting a two-step sample pretreatment in perchloric acid procedure to eliminate the need for DNA extraction. Here, we further simplified the Burton method by replacing the previous two-step perchloric acid pretreatment with a new and one-step diphenylamine reagent pretreatment. The reliability and accuracy of this simplified method were assessed by measuring the biomass of four model microorganisms: Escherichia coli, Streptomyces clavuligerus, Saccharomyces cerevisiae, and Trichoderma reesei grown in normal media or those containing solid particles. The results demonstrate that this new simplified method performs comparably to the conventional methods, such as OD₆₀₀ or the previously modified Burton method, and is much more sensitive than the dry weight method. Overall, the new method is simple, reliable, easy to perform, and generally applicable in most circumstances, and it reduces the operation time from more than 12 h (for the previously simplified Burton method) to about 2 h.     

Quantification of toxigenic Microcystis spp. in freshwaters by quantitative real-time PCR based on the microcystin synthetase A gene

A method to estimate the abundance of toxigenic Microcystis in environmental samples by using quantitative real-time PCR was developed and optimized. The basis of this method is the amplification of a highly conserved region of the mcyA gene within the microcystin synthetase gene cluster. Using this method, the average copy number of mcyA gene per cell in toxigenic Microcystis strains was estimated. The molecular markers and method developed in this study can be used to monitor toxigenic strains of Microcystis in Korean freshwaters, in which harmful cyanobacterial blooms are routinely found.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.     

Extracellular Space Volume Measured by Two-Color Pulsed Dye Infusion with Microfiberoptic Fluorescence Photodetection

The extracellular space (ECS) is the aqueous matrix surrounding cells in solid tissues. The only method to measure ECS volume fraction (α) in vivo has been tetramethylammonium iontophoresis, a technically challenging method developed more than 25 years ago. We report a simple, quantitative method to measure α by microfiberoptic fluorescence detection of a self-quenched green dye, calcein, and a reference red dye, sulforhodamine 101, after pulsed iontophoretic infusion. The idea is that the maximum increase in calcein fluorescence after iontophoresis is proportional to the aqueous volume into which the dye is deposited. We validated the method theoretically, and experimentally, using cell-embedded gels with specified α and ECS viscosity. Measurements in living mice gave α of 0.20 ± 0.01 in brain, 0.13 ± 0.02 in kidney and 0.074 ± 0.01 in skeletal muscle. The technical simplicity of the ”pulsed-infusion microfiberoptic photodetection” method developed here should allow elucidation of the relatively understudied biological roles of the ECS.     

An enzymic method for determination of the average chain lengths of glycogens and amylopectins

A new method for the determination of the average chain lengths of glycogens and amylopectins is described. The method utilizes yeast glucosidase-transferase and sweet-potato β-amylase, and was tested successfully against a number of samples of polysaccharides of known chain lengths. The method has several advantages over previous enzymic methods in that (1) it is relatively insensitive to the presence of α-amylase, (2) the enzymes required are readily available—sweet-potato β-amylase is commercially available and we describe a relatively simple method for the isolation of the yeast glucosidase-transferase, (3) the time required for the assay is short, relative to the method of Lee and Whelan (8).     

Acanthamoeba-Campylobacter Coculture as a Novel Method for Enrichment of Campylobacter Species

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10⁴ CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.     

An efficient autofluorescence method for screening Limonium bicolor mutants for abnormal salt gland density and salt secretion

With the spread of saline soils worldwide, it has become increasingly important to understand salt-tolerant mechanisms and to develop halophytes with increased salt tolerance. Limonium bicolor is a typical recretohalophyte and has a typical salt excretory structure in the epidermis called the salt gland. A method that can be used to screen a large population of L. bicolor mutants for altered salt gland density and altered salt secretion is needed but is currently unavailable. Leaves of 1-month-old L. bicolor seedlings were processed by three traditional methods [epidermal peel, nail impression, and clearing/differential interference contrast microscope (clearing/DIC) method] and a fluorescence method (fluorescence microscopic examination of cleared leaves). With the fluorescence method, the autofluorescence of salt glands under UV excitation (330–380 nm) was easily distinguished with the least labor and time. The fluorescence method was used to screen ~ 10,000 seedlings (which grew from gamma-irradiated seeds). Four mutants with reduced salt gland density and 15 with increased salt gland density were obtained. Both kinds of mutants will be useful for the isolation of genes involved in salt gland development and salt secretion in L. bicolor and other halophytes. The fluorescence method was also successfully used to observe the salt glands of Aegialitis rotundifolia and the stomata and trichomes of Arabidopsis. The fluorescence method described here will be useful for examining plant epidermal structures that have autofluorescence under UV or other wavelengths.     

Amino-terminal analysis of polypeptide using dimethylaminoazobenzene isothiocyanate

A new method for qualitative and quantitative N-terminal analysis of polypeptide using dimethylaminoazobenzene-isothiocyanate is presented. The method can recover all naturally occurring N-terminal amino acids, including asparagine, glutamine, and tryptophane in a nearly quantitative yield. Less than 1 nmol of polypeptide is required for qualitative N-terminal analysis and 5 to 10 nmol of polypeptide is used for quantitative N-terminal analysis. Applications and expected limitations of this new N-terminal method are described.     

Determination of nicotinamide N-oxide—a human excretory product

A method for the determination of nicotinamide N-oxide has been developed. It is based on the ability of the N-oxide to function as an electron acceptor in the xanthine oxidase catalyzed oxidation of xanthine. In simple mixtures the N-oxide can be converted quantitatively to nicotinamide and the latter determined by the cyanogen bromide method. The conversion is not always quantitative in complex mixtures, such as urine; an isotope dilution variation on the basic method permits the determination of the N-oxide in such situations. The basic method is applicable over the range 0.02–0.3 μmole of nicotinamide N-oxide.The new method has been used to verify the prominent excretory role of nicotinamide N-oxide in rodents. Application of the method to a study of human urines has permitted the detection of the N-oxide as an excretory metabolite in man. Only vanishingly small quantities of the N-oxide are excreted under normal conditions. However after the ingestion of 200 mg of nicotinamide, significant quantities of the N-oxide are detectable in human urine. Urine samples obtained from a number of other mammalian species contained little or no detectable nicotinamide N-oxide.     

Open-sea cultivation by transplanting young fronds of the kelp Saccharina latissima

Saccharina latissima is an economically and ecologically important native kelp. As its limited supply from wild stock cannot meet increasing current and future demands, methods for its cultivation in the ocean need to be developed. This kelp is now beginning to be farmed off the Atlantic coast of Spain using a regular method similar to the “forced cultivation” technique used with Asian kelps (kombu). Its cultivation is also a growing enterprise in other European countries. In this study, the open-sea farming of S. latissima using the transplanting method is tested on a commercial scale. This cultivation method has not been studied with kelp species outside Asian waters. The tested method includes the following steps: indoor production of seedlings, pre-culture in greenhouse tanks, and open-sea cultivation by transplanting young fronds. Results demonstrate that open-sea cultivation using transplanted young fronds is a technically and biologically viable method. The mean yield obtained (7.8 kg fresh wt per meter rope equivalent to 45.6 t fresh wt per hectare farm) is satisfactory, considering the low densities of transplanted fronds (25–30 fronds per meter rope). Moreover, these values are comparable to those reported in previous cultivations with this species, as well as in the farming of similar kelps. The transplanting method used in conjunction with the regular cultivation method has valuable practical applications for the commercial farming of S. latissima.