Analysis of the Phenotype and the Restriction Enzyme Mapping Level of Mutations Induced by the New Mutagen Glycidyl Methacrylate

Glycidyl methaerylate (GMA) is a recently recognized chemical mutagen. In order to explore the mutagenicity and mutagenic process of GMA, plasmid pBR322 was used for in vitro binding, mutant screening, and restriction enzyme mapping. The binding between GMA and DNA in vitro has been verified by means of a spectrophotometric method. When pBR322 and GMAbound pBR322 were used to transform Eschenchia coli HB101, the following results were obtained: (1) The transformation efficiency of GMA-bound pBR322 was much lower than that of pBR322 alone. (2) GMA-bound pBR322 induced phenotype changes in competent cells (i.e., tetracycline-resistance inactivation or ampicillin-resistance inactivation). There were two mutants of pBR322, Ap~RTc~S and Ap~STc~R, in the transformants and a deductive mutant Ap~STc~S in the nontranstormants. (3) All of the selected mutants were stable and heritable. (4) When restriction enzyme maps were used to analyze the mutant Ap~RTc~S, four of seven maps were changed. some sites were shifted to other resistant gene regions, for example, sites of Bgll, EcoRl, Ilindlll. Hinclll, etc., and there was a new recognition site for Hindi (252). We did not observe any DNA fragment insertion or deletion on any maps. Our results suggest that when GMA is covalently linked to the plasmid DNA, it gives rise to a premutagenic lesion of DNA that is converted in vivo into a point mutation. (C)1990 Academic Press, Inc.

Evaluation of the Method Based on Restriction Fragment Length Polymorphism Analysis as Simple Analysis Method of Lactic Acid Bacteria in Foods

Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g-1) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.

The burden of upper motor neuron involvement is correlated with the bilateral limb involvement interval in patients with amyotrophic lateral sclerosis:a retrospective observational study

Amyotrophic lateral sclerosis is a rare neurodegenerative disease characterized by the involvement of both upper and lower motor neurons.Early bilateral limb involvement significantly affects patients'daily lives and may lead them to be confined to bed.However,the effect of upper and lower motor neuron impairment and other risk factors on bilateral limb involvement is unclear.To address this issue,we retrospectively collected data from 586 amyotrophic lateral sclerosis patients with limb onset diagnosed at Peking University Third Hospital between January 2020 and May 2022.A univariate analysis revealed no significant differences in the time intervals of spread in different directions between individuals with upper motor neuron-dominant amyotrophic lateral sclerosis and those with classic amyotrophic lateral sclerosis.We used causal directed acyclic graphs for risk factor determination and Cox proportional hazards models to investigate the association between the duration of bilateral limb involvement and clinical baseline characteristics in amyotrophic lateral sclerosis patients.Multiple factor analyses revealed that higher upper motor neuron scores(hazard ratio[HR]=1.05,95%confidence interval[CI]=1.01–1.09,P=0.018),onset in the left limb(HR=0.72,95%CI=0.58–0.89,P=0.002),and a horizontal pattern of progression(HR=0.46,95%CI=0.37–0.58,P<0.001)were risk factors for a shorter interval until bilateral limb involvement.The results demonstrated that a greater degree of upper motor neuron involvement might cause contralateral limb involvement to progress more quickly in limb-onset amyotrophic lateral sclerosis patients.These findings may improve the management of amyotrophic lateral sclerosis patients with limb onset and the prediction of patient prognosis.

Model and algorithm of optimizing alternate traffic restriction scheme in urban traffic network

An optimization model and its solution algorithm for alternate traffic restriction(ATR) schemes were introduced in terms of both the restriction districts and the proportion of restricted automobiles. A bi-level programming model was proposed to model the ATR scheme optimization problem by aiming at consumer surplus maximization and overload flow minimization at the upper-level model. At the lower-level model, elastic demand, mode choice and multi-class user equilibrium assignment were synthetically optimized. A genetic algorithm involving prolonging codes was constructed, demonstrating high computing efficiency in that it dynamically includes newly-appearing overload links in the codes so as to reduce the subsequent searching range. Moreover,practical processing approaches were suggested, which may improve the operability of the model-based solutions.

Succession of Bacterial Community Inhabited Acid Mine Drainage under High Fe（Ⅱ） Concentration

To reveal the effects of Fe2＋ on bacterial communities in the early stages of minerals dissolution, two different acid mine drainage （AMD） samples were collected at Dabaoshan Mine and Shenbu Mine. Community successions of AMD niches were analyzed by Amplified Ribosomal DNA Restriction Analysis （ARDRA）, sequencing, and phylogenetic analysis in original AMD samples and their subculture under Fe2＋ concentrations. Although geochemical properties and community structures were greatly different between the two original AMD samples, bacterial community successions were still very similar under high Fe2＋ concentrations. The results showed that Acidithiobacillus ferrooxidans have competitive relationship with other bacterial species living in the AMD, including species that were also capable of oxidizing ferrous ion. A competitive relationship among different At. ferrooxidans strains likewise existed. Some of At. ferrooxidans can grow first under conditions of high ferrous ion concentration, and other At. ferrooxidans species decreased gradually and disappeared. This suggested that these species of At. ferrooxidans are most acidophilic bacteria and afford Fe3＋ to leach other metallic ion in the early stages of minerals dissolution.

Pitfalls of restriction enzyme analysis in identifying, characterizing,typing, and naming viral pathogens in the era of whole genome data, as illustrated by HAdV type 55

Restriction endonuclease analysis(REA),or restriction fragment length polymorphism(RFLP),was useful for identifying and determining the relatedness and putative identities of microbial strains(Tang et al.,1997)and for characterizing and discriminating large numbers of samples inexpensively in the past。

Novel ACTG1 mutation causing autosomal dominant non-syndromic hearing impairment in a Chinese family

γ -actin （ACTG1） gene is a cytoplasmic nonmuscle actin gene, which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea. Mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families, respectively. In this study, a novel missense mutation （c.364A〉G; p.I122V） co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls. The alteration of residue Ile122 was predicted to damage its interaction with actin-binding proteins, which may cause disruption of hair cell organization and function. These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum of ACTG1 mutations causing hearing loss.

Identification and Enumeration Method of Both Eukaryotic and Prokaryotic Microorganisms in Food Sample

The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g-1, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g-1. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g-1. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g-1) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g-1), and the other Firmicutes (3.7 × 106 MPN g-1). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g-1), Burkhokderia sp. (1.5 × 106 MPN g-1), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g-1). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.

A Simple Evaluation System for Microbial Property in Soil and Manure

Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 106 MPN g-1 dry soil), followed by γ-Proteobacteria (1.32 × 106 MPN), δ-Proteobacteria (0.006 × 106 MPN), and the other gram negative bacteria (0.006 × 106 MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 106 MPN), followed by α-Proteobacteria (26.0 × 106 MPN), β-Proteobacteria (17.1 × 106 MPN), δ-Proteobacteria (11.2 × 106 MPN), the other Firmicutes (1.71 × 106 MPN), γ-Proteobacteria (0.5 × 106 MPN), and the other gram negative bacteria (0.05 × 106 MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 106 MPN), followed by Actinobacteria (2.14 × 106 MPN), Bacillus sp. (2.14 × 106 MPN), and γ-Proteobacteria (0.35 × 106 MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candidate of a new evaluation system of soil and manure.

A New Evaluation Method for Antibiotic-Resistant Bacterial Groups in Environment

In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L-1) were included in row cattle feces (1.78 × 104 MPN g-1) and cattle feces manure (>3.84 × 104 MPN g-1). Compost originated from leftover food (>44.8 × 104 MPN g-1) and shochu lee (>320 × 104 MPN g-1) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L-1), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 104 MPN g-1), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L-1 of ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 104 MPN g-1), followed by cattle feces manure (4.19 × 104 MPN g-1), and shochu lee (0.36 × 104 MPN g-1), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.

Microbial Activity and Culturable Bacterial Diversity in Sediments of the Great Rann of Kachchh,Western India

The Great Rann of Kachchh, a vast expanse of salt desert in Western India is a unique hostile ecosystem posing an extreme environment to life forms due to high salt content, hyper-axid climate, seasonal water logging and extremes of temperature. In the virtual absence of natural vegetation, soils and sediments of Rann of Kachchh axe microbially dominated ecosystems. In the present study microbial activity and the diversity of cultivated heterotrophic bacteria were investigated in the sediments collected along a 5-m exposed section at Khadir Island in the Great Rann of Kachchh. Microbial activity （as an index of sediment enzymes） was found to be high in the middle of the section （200-280 cm）. Dehydrogenase （DHA）, substrate-induced DHA and alkaline phosphatase activities revealed the oligotrophic nature of the basal portion （320-480 cm）. Abundant bacterial isolates obtained from different depths were found to be clustered in 12 different phylogenetic groups by amplified ribosomal DNA restriction analysis. 16S rRNA gene sequencing revealed the dominant bacterial ribotypes to be affiliated to Firmicutes （Families Bacillaceae and Staphyloeoccaeeae） and Aetinobaeteria （Family Brevibaeteriaceae） with minor contribution of Proteobacteria （Families Phyllobacteriaeeae and Bartonellaceae）, pointing their endurance and adaptability to environmental stresses. Statistical analysis indicated that sediment organic carbon, salinity, total available nitrogen and total available phosphorous are most likely critical determinants of microbial activity in the Khadir Island sediments.

Phosphate Solubilizing Ability and Phylogenetic Diversity of Bacteria from P-Rich Soils Around Dianchi Lake Drainage Area of China

The phylogenetic diversity of phosphate solubilizing bacteria （PSB） distributed in P-rich soils in the Dianchi Lake drainage area of China was characterized, and the tricalcium phosphate （TCP） solubilizing activities of isolated PSB were determined. Among 1 328 bacteria isolated from 100 P-rich soil samples, 377 isolates （28.39% of the total） that exhibited TCP solubilization activity were taken as PSB. These PSB showed different abilities to solubilize TCP, with the concentrations of solubilized P in bacterial cultures varying from 33.48 to 69.63 mg L^（-1）. A total of 123 PSB isolates, with relatively high TCP solubilization activity （〉 54.00 mg L^（-1））, were submitted for restriction fragment length polymorphism （RFLP） analysis, which revealed 32 unique RFLP patterns. Based on these patterns, 62 representative isolates, one to three from each RFLP pattern, were selected for 16S rRNA sequencing. Phylogenetic analysis placed the 123 PSB into three bacterial phyla, namely Proteobacteria, Aetinobacteria and Firmicutes. Members of Proteobacteria were the dominant PSB, where 107 isolates represented by 26 RFLP patterns were associated with the genera of Burkholderia, Pseudomonas, Acinetobacter, Enterobacter, Pantoea, Serratia, Klebsiella, Leclercia, Raoultella and Cedeeea. Firmicutes were the subdominant group, in which 13 isolates were affiliated with the genera of Bacillus and Brevibacterium. The remaining 3 isolates were identified as three species of the genus Arthrobacter. This research extends the knowledge on PSB in P-rich soils and broadens the spectrum of PSB for the development of environmentally friendly biophosphate fertilizers.