Genetic differentiation and evolution of broad-leaved evergreen shrub and tree varieties of Daphniphyllum macropodum (Daphniphyllaceae).

Tree form evolution is an important ecological specialization for woody species, but its evolutionary process with adaptation is poorly understood, especially on the microevolutionary scale. Daphniphyllum macropodum comprises two varieties: a tree variety growing in a warm temperate climate with light snowfall and a shrub variety growing in a cool temperate climate with heavy snowfall in Japan. Chloroplast DNA variations and genome-wide single-nucleotide polymorphisms across D. macropodum populations and D. teijsmannii as an outgroup were used to reveal the evolutionary process of the shrub variety. Population genetic analysis indicated that the two varieties diverged but were weakly differentiated. Approximate Bayesian computation analysis supported a scenario that assumed migration between the tree variety and the southern populations of the shrub variety. We found migration between the two varieties where the distributions of the two varieties are in contact, and it is concordant with higher tree height in the southern populations of the shrub variety than the northern populations. The genetic divergence between the two varieties was associated with snowfall. The heavy snowfall climate is considered to have developed since the middle Quaternary in this region. The estimated divergence time between the two varieties suggests that the evolution of the two varieties may be concordant with such paleoclimatic change.

The evolutionary history of rice azaleas (Rhododendron tschonoskii alliance) involved niche evolution to a montane environment.

PREMISE: The formation of isolated montane geography on islands promotes evolution, speciation, and then radiation if there are ecological changes. Thus, investigating evolutionary histories of montane species and associated ecological changes may help efforts to understand how endemism formed in islands' montane floras. To explore this process, we investigated the evolutionary history of the Rhododendron tschonoskii alliance, which grows in montane environments of the Japanese archipelago and the Korean Peninsula. METHODS: We studied the five species in the R. tschonoskii alliance and 30 outgroup species, using genome-wide single-nucleotide polymorphisms and cpDNA sequences, in association with environmental analyses. RESULTS: The monophyletic R. tschonoskii alliance diverged since the late Miocene. Species in the alliance currently inhabit a cold climatic niche that is largely different from that of the outgroup species. We observed clear genetic and niche differentiations between the taxa of the alliance. CONCLUSIONS: The association of the alliance's evolution with the formation of cooler climates on mountains indicates that it was driven by global cooling since the mid-Miocene and by rapid uplift of mountains since the Pliocene. The combination of geographic and climatic isolation promoted high genetic differentiation between taxa, which has been maintained by climatic oscillations since the Quaternary.

Complete chloroplast genome of Daphne pseudomezereum var. koreana (Thymelaeaceae).

Daphne pseudomezereum A. Gray var. koreana (Nakai) Hamaya is a shrub distributed in high mountains in Japan and Korea and is used as a medicinal plant. The complete chloroplast genome of D. pseudomezereum var. koreana is 171,152 bp long with four subregions consisting of a large single-copy region (84,963 bp), a small single-copy region (41,725 bp), and a pair of inverted repeats (2739 bp). The genome includes 139 genes (93 protein-coding genes, eight rRNAs, and 38 tRNAs). Phylogenetic analyses show that D. pseudomezereum var. koreana is nested within the Daphne clade in the narrow sense and that it forms a distinct lineage.

Genetic and demographic signatures accompanying the evolution of the selfing syndrome in Daphne kiusiana, an evergreen shrub.

BACKGROUND AND AIMS: The evolution of mating systems from outcrossing to self-fertilization is a common transition in flowering plants. This shift is often associated with the 'selfing syndrome', which is characterized by less visible flowers with functional changes to control outcrossing. In most cases, the evolutionary history and demographic dynamics underlying the evolution of the selfing syndrome remain poorly understood. METHODS: Here, we characterize differences in the demographic genetic consequences and associated floral-specific traits between two distinct geographical groups of a wild shrub, Daphne kiusiana, endemic to East Asia; plants in the eastern region (southeastern Korea and Kyushu, Japan) exhibit smaller and fewer flowers compared to those of plants in the western region (southwestern Korea). Genetic analyses were conducted using nuclear microsatellites and chloroplast DNA (multiplexed phylogenetic marker sequencing) datasets. KEY RESULTS: A high selfing rate with significantly increased homozygosity characterized the eastern lineage, associated with lower levels of visibility and herkogamy in the floral traits. The two lineages harboured independent phylogeographical histories. In contrast to the western lineage, the eastern lineage showed a gradual reduction in the effective population size with no signs of a severe bottleneck despite its extreme range contraction during the last glacial period. CONCLUSIONS: Our results suggest that the selfing-associated morphological changes in D. kiusiana are of relatively old origin (at least 100 000 years ago) and were driven by directional selection for efficient self-pollination. We provide evidence that the evolution of the selfing syndrome in D. kiusiana is not strongly associated with a severe population bottleneck.

Micrococcin P2 Targets Clostridioides difficile.

Clostridioides difficile infection is a global public health threat. Extensive in vitro assays using clinical isolates have identified micrococcin P2 (MP2, 1) as a particularly effective anti-C. difficile agent. MP2 possesses a mode of action that differs from other antibiotics and pharmacokinetic properties that render it especially promising. Its time-kill studies have been investigated using hypervirulent C. difficile ribotype 027. DSS (dextran sulfate sodium)-induced in vivo mouse studies with that strain indicate that 1 is better than vancomycin and fidaxomicin. Thus, micrococcin P2 is a valuable platform to be exploited for the development of new anti-C. difficile antibiotics.

Nitro-Group-Containing Thiopeptide Derivatives as Promising Agents to Target Clostridioides difficile.

The US Centers for Disease Control and Prevention (CDC) lists Clostridioides difficile as an urgent bacterial threat. Yet, only two drugs, vancomycin and fidaxomicin, are approved by the FDA for the treatment of C. difficile infections as of this writing, while the global pipeline of new drugs is sparse at best. Thus, there is a clear and urgent need for new antibiotics against that organism. Herein, we disclose that AJ-024, a nitroimidazole derivative of a 26-membered thiopeptide, is a promising anti-C. difficile lead compound. Despite their unique mode of action, thiopeptides remain largely unexploited as anti-infective agents. AJ-024 combines potent in vitro activity against various strains of C. difficile with a noteworthy safety profile and desirable pharmacokinetic properties. Its time-kill kinetics against a hypervirulent C. difficile ribotype 027 and in vivo (mouse) efficacy compare favorably to vancomycin, and they define AJ-024 as a valuable platform for the development of new anti-C. difficile antibiotics.

Antibacterial Activity of LCB10-0200 against Klebsiella pneumoniae.

Klebsiella pneumoniae is one of the important clinical organisms that causes various infectious diseases, including urinary tract infections, necrotizing pneumonia, and surgical wound infections. The increase in the incidence of multidrug-resistance K. pneumoniae is a major problem in public healthcare. Therefore, a novel antibacterial agent is needed to treat this pathogen. Here, we studied the in vitro and in vivo activities of a novel antibiotic LCB10-0200, a siderophore-conjugated cephalosporin, against clinical isolates of K. pneumoniae. In vitro susceptibility study found that LCB10-0200 showed potent antibacterial activity against K. pneumoniae, including the beta-lactamase producing strains. The in vivo efficacy of LCB10-0200 was examined in three different mouse infection models, including systemic, thigh, and urinary tract infections. LCB10-0200 showed more potent in vivo activity than ceftazidime in the three in vivo models against the drug-susceptible and drug-resistant K. pneumoniae strains. Taken together, these results show that LCB10-0200 is a potential antibacterial agent to treat infection caused by K. pneumoniae.

The complete chloroplast genome of Aruncus dioicus var. kamtschaticus (Rosaceae).

Aruncus dioicus var. kamtschaticus is an economically important herb in the cold temperate regions of East Asia, and displays highly variable morphological features. Completed chloroplast genome of A. dioicus var. kamtschaticus isolated in Korea is 157,859 bp long with four subregions: 85,972 bp of large single copy and 19,185 bp of small single-copy regions separated by 26,351 bp of inverted repeat regions. The genome includes 131 genes (86 protein-coding genes, eight rRNAs, and 37 tRNAs). Phylogenetic analyses show that our chloroplast genome was clustered with two partial chloroplast genomes of A. dioicus.

The complete chloroplast genome of Campanula takesimana Nakai from Dokdo Island in Korea (Campanulaceae).

Completed chloroplast genome of Campanula takesimana Nakai isolated from Dokdo island in Korea is 169,719 bp long (GC ratio is 38.8%) and has four subregions: 102,381 bp of large single-copy (37.8%) and 7,750 bp of small single-copy (32.6%) regions are separated by 29,794 bp of inverted repeat (41.3%) regions including 131 genes (87 protein-coding genes, eight rRNAs, and 36 tRNAs). Phylogenetic analyses suggested that C. takesimana from Dokdo Island form a clade with C. takesimana from Ulleungdo Island and that chloroplast genomes of the two accessions are diverged.

The complete chloroplast genome of Euscaphis japonica (Thunb.) Kanitz (Staphyleaceae) isolated in Korea.

The complete chloroplast genome of Euscaphis japonica (Thunb.) Kanitz isolated in Korea is 160,606 bp long and has four subregions: 89,232 bp of large single-copy and 18,734 bp of small single-copy regions are separated by 26,320 bp of inverted repeat regions including 129 genes (84 CDS, 8 rRNAs, and 37 tRNAs) and three pseudogenes. There were 424 SNPs and 809 INDELs compared with the Chinese E. japonica, useful to develop markers for phylogeographic study of the species. Phylogenetic trees show that E. japonica, representing Crossosomatales, is nested within the Malvids clade, confirming previous studies.

New taxa of Rhododendron tschonoskii alliance (Ericaceae) from East Asia.

Three new taxa, Rhododendron sohayakiense Y.Watan. & T.Yukawa (Ericaceae), and its two varieties, var. kiusianum Y.Watan., T.Yukawa & T.Minamitani and var. koreanum Y.Watan. & T.Yukawa are described and illustrated from Japan and South Korea. They can be distinguished from each other and from the other members of the R. tschonoskii alliance, i.e. R. tschonoskii, R. tetramerum, R. trinerve and R. tsusiophyllum, through their combination of leaf size, leaf morphologies including lateral nerves on abaxial leaf surface, corolla morphologies including number of corolla lobes, style length and anther form. Phylogenetic inferences based on chloroplast DNA and genome-wide sequences revealed that each of the three new taxa is monophyletic and they further form a clade. Distributions of the three taxa are also clearly separated from each other and also from the other members of the R. tschonoskii alliance.

Pbp1-Interacting Protein Mkt1 Regulates Virulence and Sexual Reproduction in Cryptococcus neoformans.

The Mkt1-Pbp1 complex promotes mating-type switching by regulating the translation of HO mRNA in Saccharomyces cerevisiae. Here, we performed in vivo immunoprecipitation assays and mass spectrometry analyses in the human fungal pathogen Cryptococcus neoformans to show that Pbp1, a poly(A)-binding protein-binding protein, interacts with Mkt1 containing a PIN like-domain. Association of Pbp1 with Mkt1 was confirmed by co-immunoprecipitation assays. Results of spot dilution growth assays showed that unlike pbp1 deletion mutant strains, mkt1 deletion mutant strains were not resistant to heat stress compared with wild-type. However, similar to the pbp1 deletion mutant strains, the mkt1 deletion mutants exhibited both, defective dikaryotic hyphal production and reduced pheromone gene (MFα1) expression during mating. In addition, deletion of mkt1 caused attenuated virulence in a murine intranasal inhalation model. Taken together, our findings reveal that Mkt1 plays a crucial role in sexual reproduction and virulence in C. neoformans.

Removal behaviors and fouling mechanisms of charged antibiotics and nanoparticles on forward osmosis membrane.

Fouling and rejection mechanisms of both charged antibiotics (ABs) and nanoparticles (NPs) were determined using a negatively-charged polyamide thin film composite forward osmosis (FO) flat sheet membrane. Two types of ABs and NPs were selected as positively and negatively charged foulants at pH 8. The ABs did not cause significant membrane fouling, but the extent of fouling and rejection changed based on the electrostatic attraction or repulsion forces. The addition of opposite charged AB and NP resulted in a decline of the membrane flux by 11.0% but a 6.5% AB average rejection efficiency improvement. On the other hand, mixing of like-charged ABs and NPs generated repulsive forces that improved average rejection efficiency about 5.5% but made no changes in the membrane flux. In addition, NPs and ABs were mixed and tested at various concentrations and pH levels to rectify the behavior of ABs. The aggregate size and removal efficiency were observed to vary with the change in the electron double layer of the mixture. It can help to make the strategy to control the ABs in the FO process and consequently it enables the FO process to produce environmentally safe effluent.

The complete chloroplast genome sequence of Goodyera schlechtendaliana in Korea (Orchidaceae).

Goodyera schlechtendaliana is a common orchid species in East Asia, providing a case to study phylogeographic structure of understory plants in warm temperate forests. Here, we present the complete chloroplast genome of the Korean G. schlechtendaliana. Its length is 153,801 bp and it has four subregions; 82,683 bp of large-single-copy and 18,048 bp of small-single-copy regions are separated by 26,535 bp of inverted repeat regions, including 133 genes (86 protein-coding genes, eight rRNAs, and 39 tRNAs). Phylogenetic analyses suggest that the chloroplast genomic data should be useful in future phylogeographic and phylogenetic studies of Goodyera.

The complete chloroplast genome sequence of Viburnum erosum (Adoxaceae).

Viburnum erosum is a deciduous shrub distributed in eastern Asia. As part of the systematic study to understand the phylogenetic relationship of V. erosum, we present the complete chloroplast genome of V. erosum. Its length is 158,624 bp and it has four subregions: 87,060 bp of large single-copy and 18,530 bp of small single-copy regions separated by a pair of inverted repeat regions of 26,517 bp each, including 129 genes (84 protein-coding genes, 8 rRNAs, and 37 tRNAs). Phylogenetic analyses show that V. erosum is sister to Viburnum japonicum, supporting morphological affinity of the two species.

The second complete chloroplast genome sequence of the Viburnum erosum (Adoxaceae) showed a low level of intra-species variations.

We presented the second complete chloroplast genome of the plant. The length of chloroplast genome is 158,587 bp, consisting of four subregions: 87,050 bp of LSC and 18,503 bp of SSC regions separated by a pair of 26,517 bp IR regions. It includes 129 genes (84 protein-coding genes, 8 rRNAs, and 37 tRNAs). A low-level of molecular variation within Viburnum erosum was found with 16 SNPs and 49 indels. The phylogenetic tree shows that the two accessions of V. erosum are clustered with Viburnum japonicum with no resolution between the species, suggesting that chloroplast genome in these species evolve slowly.

Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic.

Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.

Mon1 Is Essential for Fungal Virulence and Stress Survival in Cryptococcus neoformans.

Mon1 is a guanine nucleotide exchange factor subunit that activates the Ypt7 Rab GTPase and is essential for vacuole trafficking and autophagy in eukaryotic organisms. Here, we identified and characterized the function of Mon1, an ortholog of Saccharomyces cerevisiae Mon1, in a human fungal pathogen, Cryptococcus neoformans. Mutation in mon1 resulted in hypersensitivity to thermal stress. The mon1 deletion mutant exhibited increased sensitivity to cell wall and endoplasmic reticulum stress. However, the mon1 deletion mutant showed more resistance to the antifungal agent fluconazole. In vivo studies demonstrated that compared to the wild-type strain, the mon1 deletion mutant attenuated virulence in the Galleria mellonella insect model. Moreover, the mon1 deletion mutant was avirulent in the murine inhalation model. These results demonstrate that Mon1 plays a crucial role in stress survival and pathogenicity in C. neoformans.

Had1 Is Required for Cell Wall Integrity and Fungal Virulence in Cryptococcus neoformans.

Calcineurin modulates environmental stress survival and virulence of the human fungal pathogen Cryptococcus neoformans Previously, we identified 44 putative calcineurin substrates, and proposed that the calcineurin pathway is branched to regulate targets including Crz1, Pbp1, and Puf4 in C. neoformans In this study, we characterized Had1, which is one of the putative calcineurin substrates belonging to the ubiquitously conserved haloacid dehalogenase ß-phosphoglucomutase protein superfamily. Growth of the had1∆ mutant was found to be compromised at 38° or higher. In addition, the had1∆ mutant exhibited increased sensitivity to cell wall perturbing agents, including Congo Red and Calcofluor White, and to an endoplasmic reticulum stress inducer dithiothreitol. Virulence studies revealed that the had1 mutation results in attenuated virulence compared to the wild-type strain in a murine inhalation infection model. Genetic epistasis analysis revealed that Had1 and the zinc finger transcription factor Crz1 play roles in parallel pathways that orchestrate stress survival and fungal virulence. Overall, our results demonstrate that Had1 is a key regulator of thermotolerance, cell wall integrity, and virulence of C. neoformans.

In Vivo Activity of LCB 01-0699, a Prodrug of LCB 01-0648, against Staphylococcus aureus.

LCB01-0648 is a novel oxazolidinone compound that shows potent antibacterial activities against most Gram-positive cocci, including the multi-drug resistant Staphylococcusaureus. In this study, in vivo activity of LCB01-0699, a LCB01-0648 prodrug, against S.aureus was evaluated in comparison with that of Linezolid. The results of the systemic infection study demonstrated that LCB01-0699 was more potent than Linezolid against methicillin-susceptible and -resistant S. aureus strains. The in vivo efficacy of LCB01-0699 against methicillin-susceptible and -resistant S. aureus strains in a skin infection model showed more potent activity than Linezolid. LCB01-0699 shows potent in vivo activity against methicillin-susceptible and -resistant S. aureus strains, suggesting that LCB01-0699 would be a novel candidate for the treatment of these infectious diseases caused by S. aureus.