Fine-tuning an aromatic ring-hydroxylating oxygenase to degrade high molecular weight polycyclic aromatic hydrocarbon.

Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) play pivotal roles in determining the substrate preferences of polycyclic aromatic hydrocarbon (PAH) degraders. However, their potential to degrade high molecular weight PAHs (HMW-PAHs) has been relatively unexplored. NarA2B2 is an RHO derived from a thermophilic Hydrogenibacillus sp. strain N12. In this study, we have identified four "hotspot" residues (V236, Y300, W316, and L375) that may hinder the catalytic capacity of NarA2B2 when it comes to HMW-PAHs. By employing structure-guided rational enzyme engineering, we successfully modified NarA2B2, resulting in NarA2B2 variants capable of catalyzing the degradation of six different types of HMW-PAHs, including pyrene, fluoranthene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene. Three representative variants, NarA2B2W316I, NarA2B2Y300F-W316I, and NarA2B2V236A-W316I-L375F, not only maintain their abilities to degrade low-molecular-weight PAHs (LMW-PAHs) but also exhibited 2 to 4 times higher degradation efficiency for HMW-PAHs in comparison to another isozyme, NarAaAb. Computational analysis of the NarA2B2 variants predicts that these modifications alter the size and hydrophobicity of the active site pocket making it more suitable for HMW-PAHs. These findings provide a comprehensive understanding of the relationship between three-dimensional structure and functionality, thereby opening up possibilities for designing improved RHOs that can be more effectively used in the bioremediation of PAHs.

A multifunctional flavoprotein monooxygenase HspB for hydroxylation and C-C cleavage of 6-hydroxy-3-succinoyl-pyridine.

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.

Blunted diurnal firing in lateral habenula projections to dorsal raphe nucleus and delayed photoentrainment in stress-susceptible mice.

Daily rhythms are disrupted in patients with mood disorders. The lateral habenula (LHb) and dorsal raphe nucleus (DRN) contribute to circadian timekeeping and regulate mood. Thus, pathophysiology in these nuclei may be responsible for aberrations in daily rhythms during mood disorders. Using the 15-day chronic social defeat stress (CSDS) paradigm and in vitro slice electrophysiology, we measured the effects of stress on diurnal rhythms in firing of LHb cells projecting to the DRN (cellsLHb→DRN) and unlabeled DRN cells. We also performed optogenetic experiments to investigate if increased firing in cellsLHb→DRN during exposure to a weak 7-day social defeat stress (SDS) paradigm induces stress-susceptibility. Last, we investigated whether exposure to CSDS affected the ability of mice to photoentrain to a new light-dark (LD) cycle. The cellsLHb→DRN and unlabeled DRN cells of stress-susceptible mice express greater blunted diurnal firing compared to stress-näive (control) and stress-resilient mice. Daytime optogenetic activation of cellsLHb→DRN during SDS induces stress-susceptibility which shows the direct correlation between increased activity in this circuit and putative mood disorders. Finally, we found that stress-susceptible mice are slower, while stress-resilient mice are faster, at photoentraining to a new LD cycle. Our findings suggest that exposure to strong stressors induces blunted daily rhythms in firing in cellsLHb→DRN, DRN cells and decreases the initial rate of photoentrainment in susceptible-mice. In contrast, resilient-mice may undergo homeostatic adaptations that maintain daily rhythms in firing in cellsLHb→DRN and also show rapid photoentrainment to a new LD cycle.

Molecular characterization of a novel narnavirus infecting the phytopathogenic fungus Botryosphaeria dothidea.

Here, a novel mycovirus, Botryosphaeria dothidea narnavirus 5 (BdNV5), was discovered in the plant-pathogenic fungus Botryosphaeria dothidea strain ZM210167-1. The BdNV5 genome sequence is 2,397 nucleotides (nt) in length and contains a putative open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) with a molecular mass of 72.77 kDa. A BLASTp search using the RdRp amino acid (aa) sequence showed that it was most similar to the RdRp of Botryosphaeria dothidea narnavirus 4 (42.35%). In a phylogenetic tree based on RdRp aa sequences, BdNV5 clustered with members of the family Narnaviridae. BdNV5 is thus a novel member of the family Narnaviridae infecting the phytopathogenic fungus B. dothidea.

Diversity of Lasiodiplodia Species Associated with Canker and Dieback in Fruit Trees in the Henan and Shandong Provinces of China.

Lasiodiplodia is a widely distributed genus that is associated with a variety of diseases in many plant species, especially fruit trees. In this study, a disease survey of fruit trees growing in 12 orchards located in the Henan and Shandong provinces of China was conducted between 2020 and 2022. The symptoms observed included stem canker, branch dieback, and gummosis. Phylogenetic analyses of internal transcribed spacer, tub2, tef1, and rpb2 sequence data combined with morphological characteristics revealed that the 19 isolates collected during the survey belonged to five documented Lasiodiplodia species, namely, Lasiodiplodia citricola, L. chiangraiensis, L. huangyanensis, L. pseudotheobromae, and L. theobromae, and two previously undescribed species, L. xinyangensis and L. ziziphi. In addition, the survey identified three novel host-pathogen interactions: L. chiangraiensis on loquat, L. citricola on apple, and L. huangyanensis on grapevine. Furthermore, the detailed phylogenic analysis indicated that four previously described Lasiodiplodia species were genetically very closely related that they would be better classified as synonyms rather than distinct species, so L. paraphysoides and L. nanpingensis should be considered synonyms of L. citricola, L. fujianensis should be a synonym of L. iraniensis, and L. henanica should be a synonym of L. huangyanensis. Pathogenicity tests confirmed that representative isolates of the two novel species and three new host-pathogen interactions identified in the current study were pathogenic to their original hosts, thereby fulfilling Koch's postulates. Similarly, all of the isolates were found to be pathogenic on four alternative hosts, although a high degree of variation in virulence was observed.

Characterization of a novel aromatic ring-hydroxylating oxygenase, NarA2B2, from thermophilic Hydrogenibacillus sp. strain N12.

Polycyclic aromatic hydrocarbons (PAHs) are harmful to human health due to their carcinogenic, teratogenic, and mutagenic effects. A thermophilic Hydrogenibacillus sp. strain N12 capable of degrading a variety of PAHs and derivatives was previously isolated. In this study, an aromatic ring-hydroxylating oxygenase, NarA2B2, was identified from strain N12, with substrate specificity including naphthalene, phenanthrene, dibenzothiophene, fluorene, acenaphthene, carbazole, biphenyl, and pyrene. NarA2B2 was proposed to add one or two atoms of molecular oxygen to the substrate and catalyze biphenyl at C-2, 2 or C-3, 4 positions with different characteristics than before. The key catalytic amino acids, H222, H227, and D379, were identified as playing a pivotal role in the formation of the 2-his-1-carboxylate facial triad. Furthermore, we conducted molecular docking and molecular dynamics simulations, notably, D219 enhanced the stability of the iron center by forming two stable hydrogen bonds with H222, while the mutation of F216, T223, and H302 modulated the catalytic activity by altering the pocket's size and shape. Compared to the wild-type (WT) enzyme, the degradation ratios of acenaphthene by F216A, T223A, and H302A had an improvement of 23.08%, 26.87%, and 29.52%, the degradation ratios of naphthalene by T223A and H302A had an improvement of 51.30% and 65.17%, while the degradation ratio of biphenyl by V236A had an improvement of 77.94%. The purified NarA2B2 was oxygen-sensitive when it was incubated with L-ascorbic acid in an anaerobic environment, and its catalytic activity was restored in vitro. These results contribute to a better understanding of the molecular mechanism responsible for PAHs' degradation in thermophilic microorganisms.IMPORTANCE(i) A novel aromatic ring-hydroxylating oxygenase named NarA2B2, capable of degrading multiple polycyclic aromatic hydrocarbons and derivatives, was identified from the thermophilic microorganism Hydrogenibacillus sp. N12. (ii) The degradation characteristics of NarA2B2 were characterized by adding one or two atoms of molecular oxygen to the substrate. Unlike the previous study, NarA2B2 catalyzed biphenyl at C-2, 2 or C-3, 4 positions. (iii) Catalytic sites of NarA2B2 were conserved, and key amino acids F216, D219, H222, T223, H227, V236, F243, Y300, H302, W316, F369, and D379 played pivotal roles in catalysis, as confirmed by protein structure prediction, molecular docking, molecular dynamics simulations, and point mutation.

Half-Sandwich Iridium(III), Rhodium(III), and Ruthenium(II) Complexes Chelating Hybrid sp2-N/sp3-N Donor Ligands to Achieve Improved Anticancer Selectivity.

The biological efficacy of half-sandwich platinum group organometallic complexes of the formula [(η5-Cpx)/(η6-arene)M(XY)Cl]0/+ (XY = bidentate ligands; Cpx = functionalized cyclopentadienyl; M = Ir, Rh, Ru, Os) has received considerable attention due to the significance of the metal center, chelating ligand, and Cpx/arene moieties in defining their anticancer potency and selectivity. With a facile access to the BIAN-derived imine-amine ligands using alkylaluminum as the reductant, we herein described the preparation and characterization of 16 half-sandwich Ir(III), Rh(III), and Ru(II) complexes chelating the hybrid sp2-N/sp3-N donor ligand. A nonplanar five-member metallacycle was confirmed by X-ray single-crystal structures of Ir1-Ir3, Ir7, Rh1, Ru1, and Ru4. The attempt to prepare imine-amido complexes using a base as the deprotonating agent led to the mixture of imine-amine complexes, within which the leaving group Cl- was displaced, and 16-electron imine-amido complexes without Cl-. The half-sandwich imine-amine complexes in this system underwent rapid hydrolysis in aqueous solution, exhibited weak photoluminescence, and showed the ability of binding to CT-DNA and BSA. The cytotoxicity of all imine-amine complexes against A549 lung cancer cell lines, HeLa cervical cancer cell lines, and 4T1 mouse breast cancer cells was determined by an MTT assay. The IC50 values of these complexes were in a range of 5.71-67.28 µM. Notably, most of these complexes displayed improved selectivity toward A549 cancer cells versus noncancerous BEAS-2B cells in comparison with the corresponding α-diimine complexes chelating the sp2-N/sp2-N donor ligand, which have been shown no selectivity in our previous report. The anticancer selectivity of these complexes appeared to be related to the redox-based mechanism including the catalytic oxidation of NADH to NAD+, reactive oxygen species (ROS) generation, and depolarization of the mitochondrial membrane. Further, inducing apoptosis of these complexes in A549 cancer cells and BEAS-2B normal cells also correlated with their anticancer selectivity, indicating the apoptosis mode of cell death in this system. In addition, these complexes could enter A549 cells via energy-dependent pathway and were able to impede the in vitro migration of A549 cells.

Potent Half-Sandwich 16-/18-Electron Iridium(III) and Ruthenium(II) Anticancer Complexes with Readily Available Amine-Imine Ligands.

The synthesis and biological evaluation of stable 16-electron half-sandwich complexes have remained scarce. We herein present the different coordination modes (16-electron or 18-electron) between half-sandwich iridium(III) complexes and ruthenium(II) complexes derived from the same amine-imine ligands chelating hybrid sp3-N/sp2-N donors. The 16-electron iridium(III) and 18-electron ruthenium(II) complexes with different counteranions were obtained and identified by various techniques. The promising cytotoxicity of these complexes against A549 lung cancer cells, cisplatin-resistant A549/DPP cells, cervical carcinoma HeLa cells, and human hepatocellular liver carcinoma HepG2 cells was observed with IC50 values ranging from 5.4 to 16.3 µM. Moreover, these complexes showed a certain selectivity (selectivity index: 2.1-3.7) toward A549 cells and BEAS-2B normal cells. The variation of metal center, counteranion, 16/18-electron coordination mode, and ligand substituents showed little influence on the cytotoxicity and selectivity of these complexes. The mechanism of action study showed that these complexes could target mitochondria, induce the depolarization of the mitochondrial membrane, and promote the generation of intracellular reactive oxygen species (ROS). Further, the induction of cell apoptosis and the perturbation of the cell cycle in the G0/G1 phase were also observed for these complexes. Overall, it seems that the redox mechanism dominated the anticancer efficacy of these complexes.

A novel bipartite negative-stranded RNA mycovirus of the order Bunyavirales isolated from the phytopathogenic fungus Fusarium sibiricum.

A novel negative-stranded RNA mycovirus was isolated from the phytopathogenic fungus Fusarium sibiricum strain AH32. This virus, tentatively named "Fusarium sibiricum coguvirus 1" (FsCV1), has a bipartite genome consisting of two RNA segments (RNA1 and RNA2). The negative-sense RNA1 is 6711 nt in length, encoding the RNA-dependent RNA polymerase (RdRp, p251) in the viral complementary (vc) strand. The ambisense RNA2 (1204 nt long) encodes two proteins from overlapping genes: the nucleocapsid protein (NP, p38) in the vc strand and a protein of unknown function (UFP, p36) in the viral (v) strand. In contrast to other Bunyavirales members, in FsCV1, the two open reading frames are separated by a long AU-rich intergenic region (IR). Sequence comparisons and phylogenetic analysis based on RdRp and NP sequences demonstrated that FsCV1 is a novel bipartite negative-stranded RNA mycovirus of the genus Coguvirus, family Phenuiviridae, order Bunyavirales.

Molecular characterization of a novel ourmia­like virus from the phytopathogenic fungus Botryosphaeria dothidea.

Here, we describe a novel ourmia-like virus, Botryosphaeria dothidea ourmia-like virus 2 (BdOLV2), derived from the phytopathogenic fungus Botryosphaeria dothidea strain ZM180192-1 infecting maize in Henan province of China. The complete genome sequence of BdOLV2 consists of a positive-sense single-stranded RNA (+ ssRNA) segment with a length of 2,532 nucleotides (nt). The sequence contains a large open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) consisting of 605 amino acids (aa) with a molecular mass of 68.59 kDa. This RdRp protein contains eight typical conserved motifs associated with ourmia-like viruses. BLASTp analysis revealed that the RdRp protein of BdOLV2 had the highest similarity (62.10%, 58.15%, and 55.75% identity, respectively) to a virus previously identified as "Botourmiaviridae sp.", Macrophomina phaseolina ourmia-like virus 2, and Macrophomina phaseolina ourmia-like virus 2-A. Phylogenetic analysis based on the RdRp aa sequence indicated that BdOLV2 is a new member of the genus Magoulivirus in the family Botourmiaviridae.

Lasiodiplodia regiae sp. nov.: A New Species Causing Canker and Dieback of Fruit Trees in China.

Canker and dieback are serious fungal diseases of woody plants that can cause huge economic losses to orchards. The purpose of this study was to classify and assess the pathogenicity of fungal species associated with canker and dieback on fruit trees growing in Henan Province, China. In total, 150 isolates of Botryosphaeriaceae were obtained from six different fruit trees exhibiting typical symptoms of stem canker, branch dieback, and gummosis. Morphological examinations and phylogenetic analysis of ITS, tef1, tub2, and rpb2 revealed two Botryosphaeriaceae species, which are Botryosphaeria dothidea and a novel species, Lasiodiplodia regiae, respectively. Using Koch's postulates, we confirmed that the different isolates of L. regiae can cause disease in their original hosts. The pathogenicity tests showed that L. regiae can cause canker, dieback, and gummosis symptoms in four different hosts, indicating a relatively wider host range. Moreover, 10 L. regiae isolates exhibited similar symptoms but different levels of virulence on shoots of peach trees under field conditions. This study demonstrated that L. regiae was a new causal agent of canker and dieback of six fruit tree species, which could be a serious risk to the orchard industry in China. Furthermore, the findings provide a foundation for further epidemiological studies and the development of management strategies.

SpyGlass in Diagnosis of Hepatocellular Carcinoma with Right Hepatic Duct Tumor Thrombus Hemorrhage: A Case Report.

Hepatocelluar carcinoma presenting as a biliary duct tumor thrombus is a relatively rare entity, with poor prognosis. The primary clinical manifestation of this disease is obstructive jaundice, which can often be misdiagnosed. A 59-year-old female patient was admitted with sudden onset of abdominal pain. Laboratory tests suggested obstructive jaundice, and enhanced magnetic resonance imaging of the upper abdomen did not show obvious biliary dilatation. Endoscopic ultrasound and endoscopic retrograde cholangiopancreatography suggested an occupying lesion in the upper bile duct. SpyGlass and biopsy finally confirmed hepatocellular carcinoma with right hepatic duct tumor thrombus hemorrhage. The SpyGlass Direct Visualization System, as an advanced biliary cholangioscopy device, showed the advantages of single-person operation as well as easy access to and visualization of the lesion.

A thermophile Hydrogenibacillus sp. strain efficiently degrades environmental pollutants polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants threatening ecosystems and human health. Here, we isolated and characterized a new strain, Hydrogenibacillus sp. N12, which is a thermophilic PAH-degrader. Strain N12 utilizes naphthalene as a sole carbon and energy source above 60°C and co-metabolizes many other PAHs as well. The metabolites were identified in the catabolism of naphthalene by gas chromatography-mass spectrometry (GC-MS) and stable isotopic analysis. Based on the identified metabolites, we proposed two possible metabolic pathways, one via salicylic acid and the other via phthalic acid. Whole-genome sequencing reveals that strain N12 possesses a small chromosome of 2.6 Mb. Combining genetic and transcriptional information, we reveal a new gene cluster for the naphthalene degradation. The genes, designated as narAaAb that are predicted to encode the alpha and beta subunits of naphthalene dioxygenase, were subsequently subcloned into Escherichia coli and the enzyme activity was detected by whole-cell transformation. Capacity to degrade several other tricyclic-PAHs was also characterized, suggesting co-existence of other constitutively expressed enzyme systems in strain N12 in addition to the naphthalene degradation gene cluster. Our study provides insights into the potential of the thermophilic PAH-degrader in biotechnology and environmental management applications.

Permeation-Enhanced Degassing Method Based on Xylem Embolism Repair and Gas Permeable Materials.

Microfluidic devices have developed a wide range of applications in the fields of biomedicine, chemistry, and analytical science. But it is easy to form and accumulate bubbles in microfluidic devices. These bubbles could decrease the detection sensitivity, cause inaccurate analysis results, and even damage the functional region of the device. Inspired by the embolism repair mechanism of angiosperms and the permeability of gas permeable materials, this work proposes a bioinspired permeation-enhanced degassing method. Bionic redundant pits are used in this method to keep bubbles from spreading between microchannels and maintain the continuity of the flow. A hydrophobic gas permeable material is used to enhance the bubble capture capability and accelerate the degassing process. This method can eliminate bubbles automatically and continuously in real time without auxiliary equipment. Compared to the bubble removal only depending on solution in water, the degassing effect of the permeation-enhanced degassing method shows about 1.6 times improvement in the same conditions, and the capability of trapping bubbles is improved by 1.33 times. In this paper, this method was integrated into a concentration gradient generator and a cell culture device. The results show that the concentration gradient generator with degassing structures can dissolve bubbles in a rapid way and reach the stability of the concentration gradient within 5-15 min. The degassing method can run for a long time and improve the cell density and cell viability of HeLa cells up to 2.64 and 1.12 times, respectively. The method has a broad application prospect in microfluidic fields including biomedical fluid processing, virus detection, and microscale reactor operation.

Formation of Iridium(III) and Rhodium(III) Amine, Imine, and Amido Complexes Based on Pyridine-Amine Ligands: Structural Diversity Arising from Reaction Conditions, Substituent Variation, and Metal Centers.

Herein, we present the different coordination modes of half-sandwich iridium(III) and rhodium(III) complexes based on pyridine-amine ligands. The pyridyl-amine iridium(III) and rhodium(III) complexes, the corresponding oxidation pyridyl-imine products, and 16-electron pyridyl-amido complexes can be obtained through the change in reaction conditions (nitrogen/adventitious oxygen atmosphere, reaction time, and solvents) and structural variations in the metal and ligand. Overall, the reaction of pyridine-amine ligands with [(η5-C5(CH3)5)MCl2]2 (M = Ir or Rh) in the presence of adventitious oxygen afforded the oxidized pyridyl-imine complexes. The possible mechanism for the oxidation of iridium(III) and rhodium(III) amine complexes was confirmed by the detection of the byproduct hydrogen peroxide. Moreover, the formation of pyridyl-amine complexes was favored when nonpolar solvent CH2Cl2 was used instead of CH3OH. The rarely reported complex with [(η5-Cp*)IrCl3] anions can also be obtained without the addition of NH4PF6. The introduction of the sterically bulky i-Bu group on the bridge carbon of the ligand led to the formation of stable 16-electron pyridyl-amido complexes. The pyridyl-amine iridium(III) and rhodium(III) complexes were also synthesized under a N2 atmosphere, and no H2O2 was detected in the whole process. In particular, the aqueous solution stability and in vitro cytotoxicity toward A549 and HeLa human cancer cells of these complexes were also evaluated. No obvious selectivity was observed for cancer cells versus normal cells with these complexes. Notably, the represented complex 5a can promote an increase in the reactive oxygen species level and induce cell death via apoptosis.

Increasing Anticancer Activity with Phosphine Ligation in Zwitterionic Half-Sandwich Iridium(III), Rhodium(III), and Ruthenium(II) Complexes.

The synthesis and biological assessment of neutral or cationic platinum group metal-based anticancer complexes have been extremely studied, whereas there are few reports on the corresponding zwitterionic complexes. Herein, the synthesis, characterization, and bioactivity of zwitterionic half-sandwich phosphine-imine iridium(III), rhodium(III), and ruthenium(II) complexes were presented. The sulfonated phosphine-imine ligand and a group of zwitterionic half-sandwich P,N-chelating organometallic complexes were fully characterized by nuclear magnetic resonance (NMR), mass spectrum (electrospray ionization, ESI), elemental analysis, and X-ray crystallography. The solution stability of these complexes and their spectral properties were also determined. Notably, almost all of these complexes showed enhanced anticancer activity against model HeLa and A549 cancer cells than the corresponding zwitterionic pyridyl-imine N,N-chelating iridium(III) and ruthenium(II) complexes, which have exhibited inactive or low active in our previous work. The increase in the lipophilic property and intracellular uptake levels of these zwitterionic P,N-chelating complexes appeared to be associated with their superior cytotoxicity. In addition, these complexes showed biomolecular interactions with bovine serum albumin (BSA). The flow cytometry studies indicated that the representative complex Ir1 could induce early-stage apoptosis in A549 cells. Further, confocal microscopy imaging analysis displayed that Ir1 entered A549 cells through the energy-dependent pathway, targeted lysosome, and could cause lysosomal damage. In particular, these complexes could impede cell migration in A549 cells.

Evidence for a novel partitivirus isolated from the entomopathogenic nematode Steinernema ceratophorum.

Nematodes are abundant, but little is known about their viruses. In this study, we report a novel partitivirus isolated from the entomopathogenic nematode species Steinernema ceratophorum, named "Steinernema ceratophorum partitivirus 1" (ScPV-1). The complete genome of ScPV-1 comprises two dsRNA segments, dsRNA1 (2352 bp) and dsRNA2 (2196 bp). Each dsRNA contains a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. The sequences of the RdRp and CP showed the highest similarity (47% and 33% identity, respectively) to Plasmopara viticola associated partitivirus 7 (PvAP-7). A multiple sequence alignment and phylogenetic analysis of the RdRp of ScPV-1 and other selected viruses indicated that ScPV-1 is a new member of the genus Betapartitivirus in the family Partitiviridae.

Synthesis and biological evaluation of zwitterionic half-sandwich Rhodium(III) and Ruthenium(II) organometallic complexes.

Herein we present the synthesis and characterization of a panel of structurally related zwitterionic piano-stool rhodium(III) and ruthenium(II) complexes. The identities of these novel complexes have been determined by NMR spectroscopy, mass spectrometry, elemental analysis and single-crystal X-ray crystallography. The stability and fluorescence property of these zwitterionic complexes were also confirmed. Zwitterionic rhodium(III) complexes Rh1-Rh4 displayed potent cytotoxic activity against A549 and HeLa human cancer cells. On the contrary, zwitterionic ruthenium(II) complexes Ru1-Ru4 presented no obvious cytotoxic activity to the test cell lines. Moreover, the trend that the introduction of fluorinated substituent and phenyl ring in the η5-CpR ring and N,N-chelating ligand, respectively, could enhance the cytotoxicity of these zwitterionic rhodium(III) complexes, were observed. The exploration of mechanism using flow cytometry displayed that the cytotoxicity of these rhodium(III) complexes was associated with the perturbation of the cell cycle and the induction of cell apoptosis. Furthermore, microscopic analysis using confocal microscopy indicated that the representative rhodium(III) complex Rh4 entered A549 cells via energy-dependent pathway and predominantly accumulated in lysosomes, thus leading to the disruption of lysosomal integrity.

Novel Subclone of Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 with Enhanced Virulence and Transmissibility, China.

We aimed to clarify the epidemiologic and clinical importance of evolutionary events that occurred in carbapenem-resistant Klebsiella pneumoniae (CRKP). We collected 203 CRKP causing bloodstream infections in a tertiary hospital in China during 2013-2017. We detected a subclonal shift in the dominant clone sequence type (ST) 11 CRKP in which the previously prevalent capsular loci (KL) 47 had been replaced by KL64 since 2016. Patients infected with ST11-KL64 CRKP had a significantly higher 30-day mortality rate than other CRKP-infected patients. Enhanced virulence was further evidenced by phenotypic tests. Phylogenetic reconstruction demonstrated that ST11-KL64 is derived from an ST11-KL47-like ancestor through recombination. We identified a pLVPK-like virulence plasmid carrying rmpA and peg-344 in ST11-KL64 exclusively from 2016 onward. The pLVPK-like-positive ST11-KL64 isolates exhibited enhanced environmental survival. Retrospective screening of a national collection identified ST11-KL64 in multiple regions. Targeted surveillance of this high-risk CRKP clone is urgently needed.

The Fluorine Effect in Zwitterionic Half-Sandwich Iridium(III) Anticancer Complexes.

The rational design by the introduction of fluorine into a compound has achieved success in the development of organic anticancer drugs. However, the fluorine effect in metal-based anticancer complexes has rarely been reported. In this contribution, we report the synthesis, characterization, chemical reactivity, and biological activity of a series of half-sandwich zwitterionic iridium(III) complexes containing different substituents in the η5-CpR ring. The molecular structures for complexes Ir1-Ir4 and Ir7 were determined by single-crystal X-ray crystallography techniques. Notably, the asymmetrically substituted fluoro complexes Ir4 and Ir6 in solution show two conformational isomers. These complexes have sufficient stability, exhibit fluorescence emission, and show potent catalytic activity in converting NADH to NAD+. The effect of the substituents in the η5-CpR ring for these zwitterionic complexes on their anticancer activity was systematically investigated. Surprisingly, the presence of fluorinated substituents gives rise to a significant increase in the anticancer activity. The lipophilicity and cellular uptake levels of these complexes appeared to be the primary factors for their cytotoxicity in this system. A microscopic mechanism study showed that the typical complex Ir4 entered A549 cancer cells through an energy-dependent pathway and was mainly located in lysosomes. Furthermore, an increase in ROS level, apoptosis induction, and cell-cycle perturbation together contribute to the anticancer potency of these zwitterionic complexes.