Search

Klebsiella pneumoniae (K. pneumoniae) is an important pathogen that can cause severe hospital- and community-acquired infections. To systematically investigate its methylation features, we determined the whole-genome sequences of 14 K. pneumoniae strains covering varying serotypes, multilocus sequence types, clonal groups, viscosity/virulence, and drug resistance. Their methylomes were further characterized using Pacific Biosciences single-molecule real-time and bisulfite technologies. We identified 15 methylation motifs [13 N6-methyladenine (6mA) and two 5-methylcytosine (5mC) motifs], among which eight were novel. Their corresponding DNA methyltransferases were also validated. Additionally, we analyzed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains, and identified differential distribution patterns of some hemi-/un-methylated GATC motifs, which tend to be located within intergenic regions (IGRs). Specifically, we characterized the in vivo methylation kinetics at single-base resolution on a genome-wide scale by simulating the dynamic processes of replication-mediated passive demethylation and MTase-catalyzed re-methylation. The slow methylation of the GATC motifs in the replication origin (oriC) regions and IGRs implicates the epigenetic regulation of replication initiation and transcription. Our findings illustrate the first comprehensive dynamic methylome map of K. pneumoniae at single-base resolution, and provide a useful reference to better understand epigenetic regulation in this and other bacterial species.

A fundamental challenge for cells is how to coordinate various biochemical reactions in space and time. To achieve spatiotemporal control, cells have developed organelles that are surrounded by lipid bilayer membranes. Further, membraneless compartmentalization, a process induced by dynamic physical association of biomolecules through phase transition offers another efficient mechanism for intracellular organization. While our understanding of phase separation was predominantly dependent on yeast and animal models, recent findings have provided compelling evidence for emerging roles of phase separation in plants. In this review, we first provide an overview of the current knowledge of phase separation, including its definition, biophysical principles, molecular features and regulatory mechanisms. Then we summarize plant-specific phase separation phenomena and describe their functions in plant biological processes in great detail. Moreover, we propose that phase separation is an evolutionarily conserved and efficient mechanism for cellular compartmentalization which allows for distinct metabolic processes and signaling pathways, and is especially beneficial for the sessile lifestyle of plants to quickly and efficiently respond to the changing environment.

Ethylmalonic encephalopathy (EE) is a rare and devastating neurodegenerative disease caused by mutations in the ETHE1 gene. It is characterized by early-onset encephalopathy, chronic diarrhea, petechiae, orthostatic acrocyanosis, and high levels of methylsuccinic, lactic, and ethylmalonic acids in body fluids. In this study, we report a patient with EE, who was identified through newborn screening, and the diagnosis was confirmed by targeted next-generation sequencing (NGS). The patient displayed recurrent petechiae, intermittent jaundice, protracted diarrhea, and extensive developmental regression. Genetic testing identified a homozygous nonsense variant, c.295C > T (p. Q99*), in the ETHE1 gene. A review of all known ETHE1 variants observed in other EE patients was conducted. This revealed the current difficulties in EE diagnosis. Besides, it also showed that patients with truncated variants of ETHE1 might exhibit pathological symptoms earlier and present more severe manifestations. Finally, a novel nonsense variant was identified, which supported and expanded our current knowledge of the variant spectrum for ETHE1. This novel variant also deepened our understanding of the genotype-phenotype associations that occur in EE patients.

Light is essential for photosynthetic organisms and is involved in the regulation of protein synthesis and degradation. The significance of light-regulated protein degradation is exemplified by the well-established light-induced degradation and repair of the photosystem II reaction center D1 protein in higher plants and cyanobacteria. However, systematic studies of light-regulated protein degradation events in photosynthetic organisms are lacking. Thus, we conducted a large-scale survey of protein degradation under light or dark conditions in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) using the isobaric labeling-based quantitative proteomics technique. The results revealed that 79 proteins showed light-regulated degradation, including proteins involved in photosystem II structure or function, quinone binding, and NADH dehydrogenase. Among these, 25 proteins were strongly dependent on light for degradation. Moreover, the light-dependent degradation of several proteins was sensitive to photosynthetic electron transport inhibitors (DCMU and DBMIB), suggesting that they are influenced by the redox state of the plastoquinone (PQ) pool. Together, our study comprehensively cataloged light-regulated protein degradation events, and the results serve as an important resource for future studies aimed at understanding light-regulated processes and protein quality control mechanisms in cyanobacteria., Graphical Abstract, Highlights • Light-/dark-regulated protein degradation events in a model Cyanobacterium were identified. • Seventy-nine proteins displayed light-regulated degradation. • Thirty-one proteins displayed dark-regulated degradation. • Multiple light-regulated protein degradation events were regulated by the redox state of the plastoquinone pool., In Brief Large-scale identification of the light/dark-regulated protein degradation events in the model cyanobacterium Synechocystis sp. PCC 6803 was conducted using quantitative proteomics. Proteins with strong degradation in light, dark, or both conditions were identified. The light-regulated degradations of multiple proteins were sensitive to photosynthetic electron transport inhibitors (DCMU and DBMIB), indicative of the redox regulation by the plastoquinone (PQ) pool in the photosynthetic electron transport chain.

Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry-SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl-HP cells and partially co-localized with BmATG6-GFP, but it almost has no significant co-localization with GFP-HaATG8. Then, the Western blot analysis demonstrated that GFP-SeATG5 conjugated with ATG12. Moreover, real-time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions.

The recent development and implementation of the advanced peak determination (APD) algorithm with MS instrument dramatically increased the sampling of low abundance features for MS/MS fragmentation. After in-depth evaluation, it is found that with APD on, many chimeric spectra are acquired through co-fragmentation of high abundance contaminants with low abundance targets, and such co-fragmentations are largely avoided when APD is off. To evaluate whether such a co-fragmentation could significantly distort the accuracy of the isobaric-labeling based quantitation of the low abundance target, a single-shot TMT experiment is performed using a two-proteome model, whereby each TMT channel contains premixed peptides from human and a cyanobacterium with a known ratio. Unexpectedly, it is found that APD does not significantly distort TMT ratios, probably because the majority of the APD-specific chimeric spectra are not identifiable. Nevertheless, a few examples of significant distortion of TMT ratios of low abundance peptides caused by APD is found through manual inspection, and suggests that APD should be off in a single-shot TMT experiment to avoid the laborious and time-costing manual inspection.

Analogues of [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt=2',6'-dimethyltyrosine), a potent μ opioid agonist peptide with mitochondria-targeted antioxidant activity were prepared by replacing Dmt with various 2',6'-dialkylated Tyr analogues, including 2',4',6'-trimethyltyrosine (Tmt), 2'-ethyl-6'-methyltyrosine (Emt), 2'-isopropyl-6'-methyltyrosine (Imt) and 2',6'-diethyltyrosine (Det). All compounds were selective μ opioid agonists and the Tmt(1)-, Emt(1) and Det(1)-analogues showed subnanomolar μ opioid receptor binding affinities. The Tmt(1)- and Emt(1)-analogues showed improved antioxidant activity compared to the Dmt(1)-parent peptide in the DPPH radical-scavenging capacity assay, and thus are of interest as drug candidates for neuropathic pain treatment.

Decoration of phytochemicals contributes to the majority of metabolic diversity in nature, whereas how this process alters the biological functions of their precursor molecules remains to be investigated. Flavones, an important yet overlooked subclass of flavonoids, are most commonly conjugated with sugar moieties by UDP-dependent glycosyltransferases (UGTs). Here, we report that the natural variation of rice flavones is mainly determined by OsUGT706D1 (flavone 7-O-glucosyltransferase) and OsUGT707A2 (flavone 5-O-glucosyltransferase). UV-B exposure and transgenic evaluation demonstrate that their allelic variation contributes to UV-B tolerance in nature. Biochemical characterization of over 40 flavonoid UGTs reveals their differential evolution in angiosperms. These combined data provide biochemical insight and genetic regulation into flavone biosynthesis and additionally suggest that adoption of the positive alleles of these genes into breeding programs will likely represent a potential strategy aimed at producing stress-tolerant plants., In contrast to flavonols, the functions of plant flavones are largely unknown. Here, the authors report the two differentially evolved glucosyltranferases (flavone 7-O-glucosyltransferase and flavone 5-O-glucosyltransferase) determine natural variation of rice flavone accumulation and UV-tolerance.

Four novel terminal modified antisense oligonucleotides (ODNs) were designed, synthesized and tested for their anti-influenza virus activity. Initial biological studies indicated that lipophilic and rimantadin emodificated Flutide exhibited more potent anti-H1N1 activity than Flutide. Among them, lipophilic modificated ODN (Flutide- I ) showed the most antiviral activity. The EC 50 value of Flutide- I for inhibiting H1N1 induced cytopathic effect (CPE) and H1N1 RNA were respectively (0.26 ± 0.16) μM and (0.11 ± 0.03) μM. The cytotoxicity of these compounds has also been assessed. No significant cytotoxicities were found for any of these compounds with the concentrations up to 20 μM.

A simple and rapid ion-pair reversed phase high-performance liquid chromatography (IP-RP-HPLC) method was developed to analyse the major impurities of lipophilic-conjugated phosphorothioate oligonucleotides (ODNs), which provided better separation performance than capillary gel electrophoresis and ion exchange chromatograph methods. The study showed that covalent conjugations of lipophilic group (docosanyl, C(22)) to ODN at 5'-termini (denoted as 5'C(22)-Flu) or 3'-termini (denoted as 3'C(22)-Flu) exhibited similar chromatographic retention behavior. Some important analytical conditions of IP-RP-HPLC, including column type, ion-pairing buffer composition, and separation temperature, were investigated for the effects on the separation of crude 5'C(22)-Flu. As expected, the method developed was successfully applied to the analysis of crude 3'C(22)-Flu and both purified products. Furthermore, the related impurities derived from the synthetic process were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrum. These MS results are of benefit to understanding the major process-related impurities in lipophilic-ODN conjugates synthesis, thereby elevating the quality of target products.

A dual solid phase extraction (SPE) pretreatment coupling with non-gel sieving capillary electrophoresis (NGCE) analysis method was established for the quantitative determination of an antisense oligodeoxynucleotide, Cantide, in rhesus monkey plasma. The conditions of SPE and the NGCE analysis were optimized. Under the optimized conditions (the SPE conditions: the pH of loading buffer was 9.0; the volumes of loading and the elution solution for the anion-exchange column were 5 mL and 3 mL, respectively. The NGCE analysis conditions: loading gel time was 30 min and the separation voltage was 24 kV), the linear dynamic range of Cantide in rhesus monkeys plasma was 1.95-250 mg/L, and the correlation coefficient (r) was more than 0. 998. The limit of quantitation was 1.95 mg/L. The intra-batch accuracies ranged from 93.38% to 100.71% with the intra-batch relative standard deviation (RSD) less than 11%. The inter-batch accuracies were from 89.46% to 103.46% with the inter-batch RSD less than 9%. The stability experiment showed that the Cantide plasma sample was stable when stored at 4 degrees C for 24 h, room temperature.for 4 h, -80 degrees C for 30 days and freeze-thaw for 2 cycles. This method was finally successfully applied to pharmacokinetic study of Cantide in rhesus monkeys.

An improved ion-pair reversed phase LC (IP-RP-LC) method was developed using triethylammonium/hexafluoroisopropanol (TEA/HFIP) buffer as the mobile phase as an alternative to capillary gel electrophoresis (CGE) and ion-exchange chromatography (IEC) for routine quality control of the synthetic 20-mer phosphorothioate oligonucleotide (PS-ODN) Cantide. The most commonly observed impurities included an oligomer with a deletion at the 5′-end (5′n-1) and a monophosphodiester (P=S/O). The method described here was sufficiently selective to simultaneously separate and identify these impurities. To the best of our knowledge, this is the first report on the separation of P=S/O analogs from PS-ODN using IP-RP-LC. Relatively high TEA concentrations and higher pH values were recommended. The separation performance was maintained even in the presence of multiple P=S/O fragments containing different PO residues. The method was successfully applied to the analysis of failure sequences of authentic crude Cantide as well as the purified product. In addition to 5′n-1 and P=S/O analogs, some minor impurities such as 3′n-1 oligomers were also partially resolved.

A novel biological sample clean-up procedure has been developed for the determination of phosphorothioate oligodeoxynucleotides (PS-ODNs) and derived metabolites in biological fluids (plasma, urine and bile) and in tissues and feces from mice and rats. This method uses a one-step C18 solid-phase extraction (SPE) for biological matrix removal, and it uses capillary gel electrophoresis (CGE) for analyte detection. The assay is specific, and its linearity is superb (r0.99) for IV-AS (a 13-mer PS-ODN) and PS19 (a 19-mer PS-ODN) in a variety of biological matrices. For both IV-AS and PS19, the precision, accuracy and absolute recovery values were found to be20%, +/-20% and 80-120%, respectively. The LODs of IV-AS and PS19 were 0.6 mg/l for plasma, 0.8 mg/l for rat urine and bile, 6 microg/g for rat tissues, and 10 microg/g for rat feces, with a signal-to-noise ratio of 3 (S/N=3). This method has been successfully applied to the analysis and quantitation of PS-ODNs in various biological samples arising from preclinical pharmacokinetic studies.

A method using anion exchange high performance liquid chromatography (AX-HPLC) and capillary gel electrophoresis (CGE) was established for the analysis of the related substances in phosphorothioate antisense oligonucleotide FT19 for quality control. The FT19 and its analogs of partial phosphodiester compounds (P=O) 1, as well as deletion sequences (n - 1) were analyzed. AX-HPLC with the DNA Pac PA-100 column (4 mm x 250 mm) was used for the separation of phosphorothioate modifications. The size of the capillary column used in the CGE was 31 cm x 100 microm with an effective length of 20 cm. It was found that full phosphorothioate and partial phosphodiester oligonucleotides of the same length were successfully separated, and the deletion sequences (n - 1) and the full-length sequence n were successfully separated in CGE. The results demonstrated that the related substances of phosphorothioate oligonucleotides can be well analyzed by AX-HPLC and CGE.

Mosquito-transmitted diseases like malaria and dengue fever are global problem and an estimated 50-100 million of dengue or dengue hemorrhagic fever cases are reported worldwide every year. The mermithid nematode Romanomermis wuchangensis has been successfully used as an ecosystem-friendly biocontrol agent for mosquito prevention in laboratory studies. However, this nematode can not undergo sex differentiation in vitro culture, which has seriously affected their application of biocontrol in the field. In this study, based on transcriptome sequencing analysis of R. wuchangensis, Rwucmab-3, Rwuclaf-1 and Rwuctra-2 were cloned and used to investigate molecular regulatory function of sex differentiation. qRT-PCR results demonstrated that the expression level of Rwucmab-3 between male and female displayed obvious difference on the 3rd day of parasitic stage, which was earlier than Rwuclaf-1 and Rwuctra-2, highlighting sex differentiation process may start on the 3rd day of parasitic stage. Besides, FITC was used as a marker to test dsRNA uptake efficiency of R. wuchangensis, which fluorescence intensity increased with FITC concentration after 16 h incubation, indicating this nematode can successfully ingest soaking solution via its cuticle. RNAi results revealed the sex ratio of R. wuchangensis from RNAi treated groups soaked in dsRNA of Rwucmab-3 was significantly higher than gfp dsRNA treated groups and control groups, highlighting RNAi of Rwumab-3 may hinder the development of male nematodes. These results suggest that Rwucmab-3 mainly involves in the initiation of sex differentiation and the development of male sexual dimorphism. Rwuclaf-1 and Rwuctra-2 may play vital role in nematode reproductive and developmental system. In conclusion, transcript sequences presented in this study could provide more bioinformatics resources for future studies on gene cloning and other molecular regulatory mechanism in R. wuchangensis. Moreover, identification and functional analysis of sex differentiation genes may clarify the sex differentiation mechanism of R. wuchangensis, which are helpful to solve the uncompleted sex differentiation problem in vitro culture and the potential large-scale field application controlling the larvae of C. quinquefasciatus, A. aegypti and A. albopictus.