Prenylated acetophenones from the roots of Calendula officinalis and their anti-inflammatory activity.

Calendula officinalis is a medicinal plant in the Asteraceae family, and it has a broad range of biological activities. In this study, we focused on the roots of C. officinalis, which have remarkable anti-inflammatory properties. By using a bioassay-guided fractionation approach, prenylated acetophenones 1 and 2-of which 1 was previously unknown-were isolated, and their structures were determined by spectroscopic analysis. Both compounds decreased lipopolysaccharide-stimulated NO production in J774.1 cells. This study could lead to the use of the Calendula roots as a natural source of inflammatory mediators.

Evaluation of NG-Test CARBA 5 for the detection of carbapenemase-producing Gram-negative bacilli.

Carbapenemase-producing Enterobacterales (CPE) pose one of the most serious antimicrobial resistance threats to public health worldwide. The outcome of CPE infection differs depending on the resistance mechanism. Therefore, rapid detection of CPE infection is essential for optimizing patient management. The carbapenem inactivation method (CIM) and modified CIM (mCIM) are standard methods for detecting CPE, but they usually require 24 h to generate results. Recently, an immunochromatographic assay, NG-Test CARBA 5, has become commercially available. It detects the five most common carbapenemase producers (KPC, IMP, NDM, VIM, and OXA-48) rapidly and accurately. We aimed to evaluate the diagnostic accuracy of NG-Test CARBA 5 for detecting carbapenemase-producing Gram-negative bacilli (CPGNB). We used 116 carbapenemase-producing strains and 48 non-carbapenemase-producing strains. Of the 116 carbapenemase-producing strains, 107 harboured genes for at least one of the five most common carbapenemases, KPC, IMP, NDM, VIM, and OXA-48-like. Forty-eight non-carbapenemase-producing strains, including carbapenem-resistant Enterobacterales, harboured genes for extended-spectrum ß-lactamases (CTX-M groups [n=25] and SHV groups [n=2]) or plasmid-mediated AmpC ß-lactamases (DHA [n=3], CMY-2 [n=2], and CFE-1 [n=1]). Antimicrobial susceptibility was tested using the agar dilution method, according to the Clinical and Laboratory Standards Institute guidelines. Of the 116 carbapenemase-producing strains, 79 were resistant to at least meropenem or imipenem. The sensitivity and specificity of the NG-Test CARBA 5 for the strains were 99.1 % (106 strains positive for 107 strains of the five most common carbapenemase producers) and 100 % (60 strains negative for other types of CPGNB [n=10] and non-CPGNB strains [n=48]), respectively. The carbapenemase-producing strain with a false-negative result produced IMP-66. The NG-Test CARBA 5 had high sensitivity and specificity for detecting carbapenemase-producing strains.

Molecular and Epidemiological Characteristics of Carbapenemase-Producing Klebsiella pneumoniae Clinical Isolates in Japan.

Carbapenemase-producing Enterobacteriaceae represent a serious public health threat worldwide. Carbapenemase genes, harbored on a transferable plasmid, have been isolated globally with distinct geographical features. Klebsiella pneumoniae, included in Enterobacteriaceae, also produces carbapenemase and often shows hypervirulence. Overlapping carbapenem resistance and hypervirulence in K. pneumoniae have been reported, but such strains have not yet been found in Japan. Here, we screened 104 carbapenemase-producing K. pneumoniae isolates collected from 37 hospitals and outpatient clinics in Japan between September 2014 and July 2015. PCR and DNA sequencing demonstrated IMP-1 in 21 isolates and IMP-6 in 83 isolates, 77 of which coharbored CTX-M-2. Most of the isolates showed low MICs toward imipenem and meropenem but high MICs toward penicillin and cephalosporins. Conjugation experiments with an Escherichia coli J53 recipient showed that most of the plasmids in IMP-6 producers were transferable, whereas only one-half of the plasmids in IMP-1 producers were transferable. PCR-based replicon typing and multiplex PCR identified five isolates belonging to the CG258 non-tonB79 cluster and no isolate belonging to the CG258-tonB79 cluster or sequence type 307 (ST307). Four K1-ST23 isolates, 10 K2-ST65 isolates, and 7 K2-ST86 isolates were detected that harbored virulence genes. The resistance genes in 85 isolates were transferable, but the virulence genes were not transferred. These results demonstrate the acquisition of IMP-type carbapenemase genes and CTX-M-type genes among hypervirulence isolates in Japan, warranting further attention and countermeasures. In this study, we have determined the molecular characteristics and epidemiology of IMP-6 producers that coharbored various CTX-M genes in Japan.IMPORTANCE Carbapenems serve as a last resort for the clinical treatment of multidrug-resistant infections. Therefore, the rapid spread of carbapenemase-producing strains represents a serious public health threat, further limiting antibiotic choices. The current findings of hypervirulent carbapenemase-producing Klebsiella pneumoniae clinical isolates in Japan demonstrate the potential broad spread and transfer of these genes, necessitating close surveillance.

Targeted gene knockin in zebrafish using the 28S rDNA-specific non-LTR-retrotransposon R2Ol.

BACKGROUND: Although most of long interspersed elements (LINEs), one class of non-LTR-retrotransposons, are integrated into the host genome randomely, some elements are retrotransposed into the specific sequences of the genomic regions, such as rRNA gene (rDNA) clusters, telomeric repeats and other repetitive sequenes. Most of the sequence-specific LINEs have been reported mainly among invertebrate species and shown to retrotranspose into the specific sequences in vivo and in vitro systems. Recenlty, 28S rDNA-specific LINE R2 elements are shown to be distributed among widespread vertebrate species, but the sequence-specific retrotransposition of R2 has never been demonstrated in vertebrates. RESULTS: Here we cloned a full length unit of R2 from medaka fish Oryzias latipes, named R2Ol, and engineered it to a targeted gene integration tool in zebrafish. By injecting R2Ol-encoding mRNA into zebrafish embryos, R2Ol retrotransposed precisely into the target site at high efficiency (98%) and was transmitted to the next generation at high frequency (50%). We also generated transgenic zebrafish carrying the enhanced green fluorescent protein (EGFP) reporter gene in 28S rDNA target by the R2Ol retrotransposition system. CONCLUSIONS: Sequence-specific LINE retrotransposes into the precise sequence using target primed reverse transcription (TPRT), possibly providing an alternative and effective targeted gene knockin method in vertebrates.

Orientation of the amino-terminal domain of ClpB affects the disaggregation of the protein.

ClpB/Hsp104 efficiently reactivates protein aggregates in cooperation with the DnaK/Hsp70 system. As a member of the AAA+ protein family (i.e. an expanded superfamily of ATPases associated with diverse cellular activities), ClpB forms a ring-shaped hexamer in an ATP-dependent manner. A protomer of ClpB consists of an N-terminal domain (NTD), an AAA+ module, a middle domain and another AAA+ module. In the crystal structures, the NTDs point to two different directions relative to other domains and are not visible in the single-particle cryo-electron microscopy reconstruction, suggesting that the NTD is highly mobile. In the present study, we generated mutants in which the NTD was anchored to other domain by disulfide cross-linking and compared several aspects of ClpB function between the reduced and oxidized mutants, using the wild-type and NTD-truncated ClpB (ClpBΔN) as references. In their oxidized form, the mutants and wild-type bind casein with a similar affinity, although the affinity of ClpBΔN for casein was significantly low. However, the extent of casein-induced stimulation of ATPase, the rate of substrate threading and the efficiency of protein disaggregation of these mutants were all lower than those of the wild-type but similar to those of ClpBΔN. These results indicate that the NTD supports the substrate binding of ClpB and that its conformational shift assists the threading and disaggregation of substrate proteins.

Compound eye development during caste differentiation in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae).

We morphologically examined postembryonic compound eye development in Reticulitermes speratus (Kolbe) to understand developmental regulation during caste differentiation. The eye primordia were shown to exist from the larval stage. The number of ommatidia and compound eye size greatly increased over the course of imaginal development. Nymphoids (second-form reproductives) possessed a developed compound eye structure on the surface of the cuticle and thick optic nerves, but individual ommatidia were not clearly discriminated. However, in the line of apterous workers and soldiers, although the outer rim of the eye was observed from second-stage workers, there were few morphological differences among instars, including ergatoids (third-form reproductives). Both nymphoids and ergatoids are slightly physogastric and have highly developed reproductive organs. These results suggest that eye development in the apterous line could be strongly arrested and that there is a weak developmental correlation between the eyes and reproductive organs in R. speratus.