In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and the validation rate of splicing isoforms, and integrated a comprehensive pipeline of splicing transcripts analysis by Iso-Seq and RNA-seq (STAIR) to identify the full-length multi-exon isoforms in rice seedling transcriptome (Oryza sativa L. ssp. japonica). STAIR discovered 11 733 full-length multi-exon isoforms, 6599 more than the SMRT Portal RS_Iso-Seq pipeline did. Of these splicing isoforms identified, 4453 (37.9%) were missed in assembled transcripts from RNA-seq reads, and 5204 (44.4%), including 268 multi-exon long non-coding RNAs (lncRNAs), were not reported in the MSU_osa1r7 annotation. Some randomly selected unreported splicing junctions were verified by polymerase chain reaction (PCR) amplification. In addition, we investigated alternative polyadenylation (APA) events in transcripts and identified 829 major polyadenylation [poly(A)] site clusters (PACs). The analysis of splicing isoforms and APA events will facilitate the annotation of the rice genome and studies on the expression and polyadenylation of AS genes in different developmental stages or growth conditions of rice.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a+) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

Several observations indicate that compatible ends of separate, yet closely linked, transposable elements (TEs) can interact in alternative transposition reactions. First, pairs of TEs cause chromosome breaks with frequencies inversely related to the intertransposon distance. Second, some combinations of two TEs produce complex rearrangements that often include DNA adjacent to one or both elements. In pairs of TEs in direct orientation, alternative reactions involving the external ends of the two TEs should lead to the transposition of a macrotransposon consisting of both elements plus the intervening chromosomal segment. Such macrotransposons have been hypothesized previously based on deletions, but no macrotransposon insertions have been recovered. To detect macrotransposition, we have analyzed heritable chromosomal rearrangements produced by a chromosome-breaking pair of Ac and Ds elements situated 6.5 kb apart in direct orientation in a part of the maize (Zea mays) genome dispensable for viability. Here, we show that the postulated macrotransposon can excise and reinsert elsewhere in the genome. In addition, this transposon pair produces other complex rearrangements, including deletions, inversions, and reshuffling of the intertransposon segment. Thus, closely linked TE pairs, a common transposition outcome in some superfamilies, are adept at restructuring chromosomes and may have been instrumental in reshaping plant genomes.

Transcriptional dynamic in response to environmental and developmental cues are fundamental to biology, yet many mechanistic aspects are poorly understood. One such example is fungal plant pathogens, which use secreted proteins and small molecules, termed effectors, to suppress host immunity and promote colonization. Effectors are highly expressed in planta but remain transcriptionally repressed ex planta, but our mechanistic understanding of these transcriptional dynamics remains limited. We tested the hypothesis that repressive histone modification at H3-Lys27 underlies transcriptional silencing ex planta, and that exchange for an active chemical modification contributes to transcription of in planta induced genes. Using genetics, chromatin immunoprecipitation and sequencing and RNA-sequencing, we determined that H3K27me3 provides significant local transcriptional repression. We detail how regions that lose H3K27me3 gain H3K27ac, and these changes are associated with increased transcription. Importantly, we observed that many in planta induced genes were marked by H3K27me3 during axenic growth, and detail how altered H3K27 modification influences transcription. ChIP-qPCR during in planta growth suggests that H3K27 modifications are generally stable, but can undergo dynamics at specific genomic locations. Our results support the hypothesis that dynamic histone modifications at H3K27 contributes to fungal genome regulation and specifically contributes to regulation of genes important during host infection.

Preimplantation genetic screening (PGS) detects chromosomal aneuploidy from DNA extracted from trophectodermal biopsy of the embryos before implantation. Although a controlled study showed no difference in pregnancy rates between this invasive cell biopsy technique and a non-biopsied control group, the potential long-term damage by the current PGS method has not be completely ruled out. We therefore tested a less-invasive protocol which utilizes spent culture medium combining with blastocoel fluid (ECB) to assess chromosomal aneuploidy. We compared the new protocol with the currently employed trophectodermal biopsy method against chromosomal information obtained from the remaining embryo. We found that the new technique generated information about aneuploidy that was not entirely identical to obtained from the biopsied trophectoderm or the remaining embryo. As the origins of the DNA extracted from the three sample types were not the same, the significance and interpretation of each result would have its own meaning. The possible implications derived from the ECB results as well as those from cell biopsy were discussed. The effectiveness of this new approach in selecting the best embryo for uterine implantation awaits further long term evaluation.

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species, and this coevolution processes is greatly driven by directional selection from host plants.

Long-term ketamine abuse is known to affect the lower urinary tract and produce symptoms of cystitis. However, the pathophysiology and causative mechanism of the changes in bladder function remain unclear. The present study aimed to investigate the existence of ketamine-induced cystitis in a rat model and characterize the underlining mechanisms. Rats were assigned to blank control, normal saline (NS), low-dose ketamine (LK, 5 mg/kg), and high-dose ketamine (HK, 50 mg/kg) groups. The two experimental groups received ketamine hydrochloride daily for 16 weeks. All rats were housed individually for assessment of urinary frequency and urine volume. Urinary biomarkers were measured at different time points. Rat bladders were excised for histopathology, immunohistochemistry, and western blot analysis. Ketamine-treated rats had increased urinary frequency compared to NS-treated rats at Week 16. Urinary nitric oxide and antiproliferative factor levels were increased in ketamine-treated rats within the first 30 h after administration. After long-term ketamine administration, urinary glycoprotein GP51 and potassium levels were decreased in the HK and LK groups compared to the NS group. Ketamine-treated rats showed thickened bladder epithelial layer, increased expression of inducible nitric oxide synthase and occludin, and decreased expression of zonula occludens-1 in the bladder wall. Ketamine, or its urinary metabolites, disrupted the proliferation of bladder epithelial cells, resulting in defected bladder epithelial barrier. Subsequent leakage of urinary potassium causes a stress response in the bladder and provokes cystitis.

c Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from Streptomyces avermitilis G8-17, an AVM industrial strain. In MHJ1011, the native aveA1 gene was seamlessly replaced with milA1 from Streptomyces hygroscopicus. The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs.A vermectins (AVMs) are a series of 16-membered macrocyclic lactone derivatives with potent anthelmintic and insecticidal properties (1, 2). Ivermectin (IVM), a hydrogenated product of AVM B1, has been one of the best-selling antiparasitics since 1981 (3). However, as insect resistance to AVM (4) and IVM (5-7) is on the rise, alternatives or new products with enhanced potency and expanded spectra of activity are urgently needed.Milbemycins (MBMs) are another group of 16-membered macrolides that share similar structures with AVMs. The insecticidal activity of milbemectin (a mixture of MBMs A3 and A4) against some parasites is higher than those of AVM and IVM, while the toxicity is significantly lower (8).The structural differences among AVM, IVM, and MBM are in C-25, C-22-23, and C-13 ( Fig. 1) (9). IVM differs from AVM in C-22-23 (the former has a saturated bond, whereas the latter has a double bond in that position), but its toxicity is lower than that of AVM (10), implying that the single bond in C-22-23 should be the better structure in terms of safety. The major structural difference between IVM and MBM is a bisoleandrosyloxy substituent attached at C-13 of IVM, whereas that position is unsubstituted in MBM. Also, there are different alkyl substituents at C-25: in IVM, the substituent can be isopropyl or secondary butyl, while in MBM, it can be methyl or ethyl. However, MBM is more potent against some parasites and is safer than IVM, suggesting that one or both of the moieties in C-25 and C-13 of MBM is (are) the better structure(s) in these ways than that (those) of IVM. Another conclusion is that the two oleandroses in C-13 are important to the antiparasitic activity of IVM, since their removal will reduce the activity significantly, about 30-fold (10). Based on the curr...