Mitogenic andadjuvant activities of polysaccharides from the cellular slime mold, Dictyostelium discoideum NC-4.

Cell surface and extracellular polysaccharide fractions obtained from Dictyostelium discoideum NC-4 cultured in bacteria-free medium showed strong B-cell mitogenic activities. Upon periodate treatment of the extra-cellular polysaccharide fraction this activity completely disappeared. The extracellular polysaccharide fraction could also enhance the antibody response in vitro against sheep red blood cells.     

Premature induction of amylase in pancreas and parotid gland of growing rats by dexamethasone

Studies were made on changes in the contents of α-amylase (EC 3.2.1.1) in the pancreas and parotid gland of rats during postnatal devlopment, on the premature induction of this enzyme by hormones and on the existence of specific glucocorticoid receptors in these tissues.The amylase content in the pancreas increased from the 9th day after birth and reached the adult level on the 28th day, its content in the parotid gland increased rapidly from the 16th to 28th day after birth and then rose more gradually to the adult level.Injection of dexamethasone into rats 6–8 days after birth induced increase in the amylase of the pancreas but not the parotid gland. However, injection of dexamethasone into weanling rats 21–23 days after birth resulted in precocious induction of amylase in both tissues.Specific glucocorticoid receptors were detectable in the parotid gland of rats from 6 days after birth but were almost undetectable in the pancreas until adolescence.     

Experession and integration of genes introduced into highly synchronized plant protoplasts

Summary Chimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G₂, M and G₁ phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.     

Sampling Strategies in Antimicrobial Resistance Monitoring: Evaluating How Precision and Sensitivity Vary with the Number of Animals Sampled per Farm

Because antimicrobial resistance in food-producing animals is a major public health concern, many countries have implemented antimicrobial monitoring systems at a national level. When designing a sampling scheme for antimicrobial resistance monitoring, it is necessary to consider both cost effectiveness and statistical plausibility. In this study, we examined how sampling scheme precision and sensitivity can vary with the number of animals sampled from each farm, while keeping the overall sample size constant to avoid additional sampling costs. Five sampling strategies were investigated. These employed 1, 2, 3, 4 or 6 animal samples per farm, with a total of 12 animals sampled in each strategy. A total of 1,500 Escherichia coli isolates from 300 fattening pigs on 30 farms were tested for resistance against 12 antimicrobials. The performance of each sampling strategy was evaluated by bootstrap resampling from the observational data. In the bootstrapping procedure, farms, animals, and isolates were selected randomly with replacement, and a total of 10,000 replications were conducted. For each antimicrobial, we observed that the standard deviation and 2.5–97.5 percentile interval of resistance prevalence were smallest in the sampling strategy that employed 1 animal per farm. The proportion of bootstrap samples that included at least 1 isolate with resistance was also evaluated as an indicator of the sensitivity of the sampling strategy to previously unidentified antimicrobial resistance. The proportion was greatest with 1 sample per farm and decreased with larger samples per farm. We concluded that when the total number of samples is pre-specified, the most precise and sensitive sampling strategy involves collecting 1 sample per farm.     

Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants’ genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites’ drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.     

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

     

Membrane properties of amacrocyclic tetraether bisphosphatidylcholine lipid: Effect of a single membrane-spanning polymethylene cross-linkage between two head groups of ditetradecylphosphatidylcholine membrane

The plasma membranes of archaea are abundant in macrocyclic tetraether lipids that contain a single or double long transmembrane hydrocarbon chains connecting the two glycerol backbones at both ends. In this study, a novel amacrocyclic bisphosphatidylcholine lipid bearing a single membrane-spanning octacosamethylene chain, 1,1’-O-octacosamethylene-2,2′-di-O-tetradecyl-bis-(sn-glycero)-3,3′-diphosphocholine (AC-(di-O-C14PC)₂), was synthesized to elucidate effects of the interlayer cross-linkage on membrane properties based on comparison with its corresponding diether phosphatidylcholine, 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DTPC), that forms bilayer membrane. Several physicochemical techniques demonstrated that while AC-(di-O-C14PC)₂ monolayer, which adopts a particularly high-ordered structure in the gel phase, shows remarkably high thermotropic transition temperature compared to DTPC bilayer, the fluidity of both phospholipids above the transition temperature is comparable. Nonetheless, the fluorescent dye leakage from inside the AC-(di-O-C14PC)₂ vesicles in the fluid phase is highly suppressed. The origin of the membrane properties characteristic of AC-(di-O-C14PC)₂ monolayer is discussed in terms of the single long transmembrane hydrophobic linkage and the diffusional motion of the lipid molecules.