DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

Seroprevalence of Bartonella henselae,Toxoplasma gondii,FIV and FeLV infections in domestic cats in Japan

Seroprevalence of Bartonella henselae, Toxoplasma gondii, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was investigated in 1,447 domestic cats derived from the north (Hokkaido) to the south (Okinawa) prefectures in Japan. Of the cats investigated, 8.8% (128/1,447) were seropositive to B. henselae, 5.4% (78/1,447) to T. gondii, 9.8% (107/1,088) to FIV, and 2.9% (32/1,088) to FeLV, respectively. For B. henselae infection, the positive rate varied from 11.5% in cats of 1 to <2 years old to 7.2% in those over 3 years old. Outdoor cats showed higher positive rate (14.5%) than that (7.0%) in indoor ones. The rate (13.5%) in flea-infested cats was significantly higher than that (7.4%) in flea-negative cats. The positive rates in southern and urban sites were more likely to be higher than those in northern and suburban sites, suggesting that warm and humid environments, density of cat population, and raising status, including hygienic condition and flea infestation in cats may correlate to higher seroprevalence of B. henselae infection. For T. gondii, FIV and FeLV infections, the seroprevalence also tended to be higher in outdoor, flea-infested cats and advanced age groups. For FIV infection, the positive rates in male (14.3%) and outdoor cats (15.0%) were significantly higher than those in female (5.0%) and indoor cats (4.6%). On the other hand, no significant difference in seropositivities was observed in FeLV and T. gondii infections concerning to both genders and raising status.     

The difference in citric acid-induced cough in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

Cough elicitation and major physiological factors influencing cough occurrence were investigated in congenitally bronchial-hypersensitive (BHS) and -hyposensitive (BHR) guinea pigs exposed to citric acid (0.3 M) aerosol for 10 min. The number of cough in BHS was significantly larger than in BHR, while the latency to cough in BHS was significantly shorter than in BHR. Pretreatment with atropine (0.2%), lidocaine (2%) or salbutamol (0.1%) aerosol and desensitization of C-fibers with capsaicin (100 mg/kg) decreased the cough numbers in both BHS and BHR. The salbutamol, atropine and capsaicin pretreatments prolonged the cough latency in BHS, but only salbutamol prolonged the latency in BHR. After salbutamol pretreatment all BHR guinea pigs exhibited cough, while 66.7% of BHS guinea pigs exhibited it. Vagal blocking by atropine suppressed coughing in both BHS and BHR. Only a small number (33.3%) of BHR guinea pigs and no BHR guinea pigs exhibited a cough response after capsaicin and lidocaine pretreatment whereas many BHS guinea pigs still produced cough after such pretreatment. The present study demonstrated that the cough responsiveness to citric acid aerosol was significantly higher in BHS than in BHR. It was revealed that airway smooth muscle contraction and functional and/or morphological development of airway nervous receptors, especially C-fiber endings, contributed to aggravation of coughing in BHS.     

Effects of Protoceratium reticulatum yessotoxin on feeding rates of Acartia hudsonica: A bioassay using artificial particles coated with purified toxin

Paralytic shellfish toxins produced by dinoflagellates are known to deter copepod grazing. Dinoflagellate species, including Protoceratium reticulatum, also produce disulfated polyether yessotoxins that were previously referred to as diarrheic shellfish toxins. However, the role of yessotoxins in predator–prey relationships is not yet clear. In the present study, the effects of purified yessotoxin (YTX) on feeding activities of Acartia hudsonica (Copepoda, Calanoida) were experimentally investigated. Polystyrene fluorescent microspheres (10 μm in diameter) colored bright blue or yellow-green were coated with cell extracts of P. reticulatum that do not produce yessotoxins. The bright blue microspheres were further coated with YTX, and the yellow-green microspheres were used as the reference. The microspheres were then given to the copepods separately or in combination to measure clearance rates and feeding selectivity. A. hudsonica was found to feed on the yellow-green microspheres without YTX at twice the rate of the bright blue microspheres with YTX. We also confirmed that microsphere color per se did not affect the feeding rates. The bright blue microspheres adsorbed 1.8–43.3 pg of YTX per microsphere, which is similar to the cell-specific yessotoxin content of toxic P. reticulatum found in natural environments. These results suggest that production of yessotoxin is advantageous for P. reticulatum by deterring predation by copepods.     

Crystallization and preliminary X-ray crystallographic analysis of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin

The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to 2.8 A resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to 2.2 A have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or I212121) with unit-cell parameters of a = 63.7, b = 124.5, and c = 126.3 A. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) is 2.71 A3 Da-1 and solvent content is 54.6%.     

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) MRM assay for stage-related proteins upon non-metastatic lung adenocarcinoma

A preceding paper suggested 81 candidates of stage-specifically expressed proteins for either stage IA or IIIA by global shotgun proteomics and spectral counting. Six proteins, a subset of these proteins, were chosen for a further verification study since they are potentially soluble and/or secretory, which nature is convenient for detecting them in blood in clinical practice. The multiple-reaction monitoring (MRM) quantitative analysis suggested that napsin-A and anterior gradient protein 2 homolog (hAG-2) out of the 6 candidates would be useful for determining stage IA or IIIA and are related to metastasis. In the study we noted that stage IIIA patients with better outcome showed napsin-A profiles similar to that of stage IA patients. We therefore examined 14 additional patients for analysis, which contained the IA-stage patients of poorer outcome and the IIIA-stage patients of better outcome. The MRM analysis of napsin-A for all patients suggests that napsin-A contents correlate with better outcome in stage IA. This and discovery studies demonstrate that direct isolation of tumor cells alone by laser microdissection (LMD) greatly reduces complexity on comprehensive analyses, and that MRM mass spectrometry using the endogenous internal standard is a feasible technology for quantitative verification of target proteins in formalin-fixed paraffin embedded (FFPE) tissues.     

Competitive abilities and distribution ranges of four asexual Daphnia pulex lineages and Daphnia mitsukuri in Eurasian continental islands

Daphnia pulex and D. mitsukuri are morphologically similar and distributed in small lowland Japanese lakes. Daphnia pulex in Japan reproduces by obligate parthenogenesis and is composed of four different lineages (JPN1–JPN4) with North American origins. Although this species is found throughout Japan, JPN1 has the largest distribution range, followed by JPN2. Daphnia mitsukuri is a putatively endemic species that is now rarely found in Japan. Since Daphnia share the same algal food, the difference in distribution ranges among the four asexual D. pulex lineages and D. mitsukuri may be caused by exploitative competition for algal food. To examine this possibility, we measured the threshold food concentration (TFC) at zero-net growth rate and the food concentration required for 50% of the individuals to survive (FC50). In addition, we examined the effects of temperature on the somatic growth rate (SGR) and the mortality rates of four asexual D. pulex lineages and D. mitsukuri. The experiments showed that TFC was lowest in D. pulex JPN1 and highest in D. mitsukuri, supporting the idea that the largest distribution range of JPN1 is due to its superiority in inter-lineage competition, and that the distribution area of D. mitsukuri is reduced through exploitative competition with D. pulex. However, although JPN2 was found in relatively large areas, it had higher TFC than JPN3 and JPN4, which appear in very limited areas. These results clearly showed that the difference in distribution ranges among D. pulex lineages are not necessarily determined by their competitive superiority alone.     

Structural conservation of the transposon Tam3 family in Antirrhinum majus and estimation of the number of copies able to transpose

We have investigated the organization of the transposon Tam3 family in Antirrhinum majus. Genomic hybridization experiments and characterization of 40 independent Tam3 clones isolated from an A. majus plant revealed that the Tam3 family is quite conserved and the copy sizes are uniform. We did not find any copy with a deleted internal sequence, unlike what is usually observed in other transposons. This exceptionally conserved structure of the Tam3 family was confirmed by PCR and sequencing analyses. Sequencing analysis identified eight copies with sequences completely identical to that of the Tam3 transposase gene. These results suggested that a considerable number of autonomous Tam3 copies are present in the genome of A. majus. Among 24 copies which are surrounded by single copy regions of the genome, 14 copies are present as specific insertions in the line which we used, but absent in other lines. These copies are therefore predicted to be movable. If this ratio is the same for all Tam3 copies in a genome, then a maximum of 60% of the copies are estimated to be movable in the genome. The relatively high frequency of gene tagged by Tam3 might reflect the large number of movable copies in the genome.