Redox‐triggered chiroptical switching activity of ruthenium(III)‐bis‐(β‐diketonato) complexes bearing a bipyridine‐helicene ligand

The charged, electroactive bipyridine‐helicene‐ruthenium(III) complex [ 4 ] ^{. +},PF₆^? has been prepared from 3‐(2‐pyridyl)‐4‐aza[6]helicene and a Ru‐bis‐(β‐diketonato)‐bis‐acetonitrile precursor (β‐diketonato: 2,2,6,6‐tetramethyl‐3,5‐heptanedionato). Its chiroptical properties (electronic circular dichroism and optical rotation) were studied both experimentally and theoretically and suggest the presence of 2 diastereoisomers, namely (P,Δ)‐ and (P,Λ)‐[ 4 ] ^{. +},PF₆^? (denoted jointly as (P,Δ*)‐[ 4 ] ^{. +},PF₆^?) and their mirror‐images (M,Λ)‐ and (M,Δ)‐[ 4 ] ^{. +},PF₆^? ((M,Δ*)‐[ 4 ] ^{. +},PF₆^?). The electrochemical reduction of (P,Δ*)‐[ 4 ] ^{. +},PF₆^? to neutral complex (P,Δ*)‐ 4 was performed and revealed strong changes in the UV‐vis and electronic circular dichroism spectra. A reversible redox‐triggered chiroptical switching process was then achieved.     

Direct observation of stepped proteolipid ring rotation in E. coli F₀F₁-ATP synthase

Although single‐molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F_oF₁‐ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F_o has been particularly hampered by signal‐to‐noise and time resolution. Here, we show the presence of a transient dwell between F_o subunits a and c by improving the time resolution to 10 μs at unprecedented S/N, and by using Escherichia coli F_oF₁ embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 μs to form and 175 μs to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c‐ring to a limit of ～36° while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F_o motor where c‐ring rotational diffusion is limited to 36°.     

Synthesis and chiroptical properties of organometallic complexes of helicenic N‐heterocyclic carbenes

Novel [4, 6]helicenes ( 4a,b ) bearing a fused imidazolium unit have been prepared from [4, 6]helicene‐2,3‐di‐n‐propyl‐amines 3a,b . The in situ formation of N‐heterocyclic carbene (NHC) derivatives followed by their complexation to iridium(I) or rhodium(I) gave access to complexes 1a , 1′a , and 1b , containing mono‐coordinated helicene‐NHC, chloro and COD (COD = 1,5‐cyclooctadiene) ligands. Ir and Rh complexes 1a and 1′a were characterized by X‐ray crystallography. HPLC and NMR analyses showed that Ir(I) complex 1b existed as a mixture of two diastereomeric complexes corresponding to enantiomeric pairs M‐(?)/P‐(+)‐ 1b ¹ and M‐(?)/P‐(+)‐ 1b ² which differ by the position of COD through space. The chiroptical properties (electronic circular dichroism and optical rotation) of the four stereoisomers were measured. These complexes were also tested as catalysts in a transfer hydrogenation reaction.     

Overproduction of PGR5 enhances the electron sink downstream of photosystem I in a C4 plant,Flaveria bidentis

C₄ plants can fix CO₂ efficiently using CO₂‐concentrating mechanisms (CCMs), but they require additional ATP. To supply the additional ATP, C₄ plants operate at higher rates of cyclic electron transport around photosystem I (PSI), in which electrons are transferred from ferredoxin to plastoquinone. Recently, it has been reported that the NAD(P)H dehydrogenase‐like complex (NDH) accumulated in the thylakoid membrane in leaves of C₄ plants, making it a candidate for the additional synthesis of ATP used in the CCM. In addition, C₄ plants have higher levels of PROTON GRADIENT REGULATION 5 (PGR5) expression, but it has been unknown how PGR5 functions in C₄ photosynthesis. In this study, PGR5 was overexpressed in a C₄ dicot, Flaveria bidentis. In PGR5‐overproducing (OP) lines, PGR5 levels were 2.3‐ to 3.0‐fold greater compared with wild‐type plants. PGR5‐like PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1), which cooperates with PGR5, increased with PGR5. A spectroscopic analysis indicated that in the PGR5‐OP lines, the acceptor side limitation of PSI was reduced in response to a rapid increase in photon flux density. Although it did not affect CO₂ assimilation, the overproduction of PGR5 contributed to an enhanced electron sink downstream of PSI.     

Orthocaspases are proteolytically active prokaryotic caspase homologues: the case of Microcystis aeruginosa

Caspases are a family of cysteine‐dependent proteases known to be involved in the process of programmed cell death in metazoans. Recently, cyanobacteria were also found to contain caspase‐like proteins, but their existence has only been identified in silico up to now. Here, we present the first experimental characterisation of a prokaryotic caspase homologue. We have expressed the putative caspase‐like gene MaOC1 from the toxic bloom‐forming cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli. Kinetic characterisation showed that MaOC1 is an endopeptidase with a preference for arginine in the P1 position and a pH optimum of 7.5. MaOC1 exhibited high catalytic rates with the k_cat/K_M value for Z‐RR‐AMC substrate of the order 10⁶ M^?1 s^?1. In contrast to plant or metazoan caspase‐like proteins, whose activity is calcium‐dependent or requires dimerisation for activation, MaOC1 was activated by autocatalytic processing after residue Arg219, which separated the catalytic domain and the remaining 55 kDa subunit. The Arg219Ala mutant was resistant to autoprocessing and exhibited no proteolytic activity, confirming that processing of MaOC1 is a prerequisite for its activity. Due to their structural and functional differences to other known caspase‐like proteins, we suggest to name these evolutionary primitive proteins orthocaspases.     

The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu‐detoxification and Cu delivery for cytochrome c oxidase (cbb₃‐Cox) assembly depend on two distinct Cu‐exporting P_1B‐type ATPases. The low‐affinity CopA is suggested to export excess Cu and the high‐affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb₃‐Cox biogenesis. In most organisms, CopA‐like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI‐like ATPases is unknown. Here we identified a CopZ‐like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox‐sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu‐sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb₃‐Cox activity is reduced, suggesting that CopZ not only supplies Cu to P_1B‐type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI‐CopZ protein complex in native R. capsulatus membranes.     

DNA methylation regulates gabrb2 mRNA expression: developmental variations and disruptions in l‐methionine‐induced zebrafish with schizophrenia‐like symptoms

Single nucleotide polymorphisms (SNPs) in the human type A gamma‐aminobutyric acid (GABA) receptor β₂ subunit gene (GABRB2) have been associated with schizophrenia and quantitatively correlated with mRNA expression in the postmortem brain tissue of patients with schizophrenia. l ‐Methionine (MET) administration has been reported to cause a recrudescence of psychotic symptoms in patients with schizophrenia, and similar symptoms have been generated in MET‐induced mice. In this study, a zebrafish animal model was used to evaluate the relationship between the gabrb2 mRNA expression and its promoter DNA methylation in developmental and MET‐induced schizophrenia‐like zebrafish. The results indicated developmental increases in global DNA methylation and decreases in gabrb2 promoter methylation in zebrafish. A significant increase in gabrb2 mRNA levels was observed after GABA was synthesized. Additionally, the MET‐triggered schizophrenia‐like symptoms in adult zebrafish, involving social withdrawal and cognitive dysfunction analyzed with social interaction and T‐maze behavioral tests, were accompanied by significantly increased DNA methylation levels in the global genome and the gabrb2 promoter. Furthermore, the significant correlation between gabrb2 mRNA expression and gabrb2 promoter methylation observed in the developmental stages became non‐significant in MET‐triggered adult zebrafish. These findings demonstrate that gabrb2 mRNA expression is associated with DNA methylation varies by developmental stage and show that these epigenetic association mechanisms are disrupted in MET‐triggered adult zebrafish with schizophrenia‐like symptoms. In conclusion, these results provide plausible epigenetic evidence of the GABA_A receptor β₂ subunit involvement in the schizophrenia‐like behaviors and demonstrate the potential use of zebrafish models in neuropsychiatric research.     

PIGMENT SIGNATURES AND PHYLOGENETIC RELATIONSHIPS OF THE PAVLOVOPHYCEAE (HAPTOPHYTA)1

Variations in the HPLC‐derived pigment composition of cultured Pavlovophyceae (Cavalier‐Smith) Green et Medlin were compared with phylogenetic relationships inferred from 18S rDNA sequencing, morphological characteristics, and current taxonomy. The four genera described for this haptophyte class (Diacronema Prauser emend. Green et Hibberd, Exanthemachrysis Lepailleur, Pavlova Butcher, and Rebecca Green) were represented by nine different species (one of which with data from GeneBank only). Chlorophylls a, c₁, c₂ and MgDVP (Mg‐[3,8‐divinyl]‐phytoporphyrin‐13²‐methylcarboxylate) and the carotenoids fucoxanthin, diadinoxanthin, diatoxanthin, and β,β‐carotene were detected in all cultures. Species only differed in the content of an unknown (diadinoxanthin‐like) xanthophyll and two polar chl c forms, identified as a monovinyl (chl c₁‐like) and a divinyl (chl c₂‐like) compound. This is the first observation of the monovinyl form in haptophytes. Based on distribution of these two chl c forms, species were separated into Pavlovophyceae pigment types A, B, and C. These pigment types crossed taxonomic boundaries at the generic level but were in complete accordance with species groupings based on molecular phylogenetic relationships and certain ultrastructural characteristics (position and nature of pyrenoid, stigma, and flagella). These results suggest that characterization of the pigment signature of unidentified culture strains of Pavlovophyceae can be used to predict their phylogenetic affinities and vice versa. Additional studies have been initiated to evaluate this possibility for the haptophyte class Prymnesiophyceae.     

WD40‐REPEAT 5a functions in drought stress tolerance by regulating nitric oxide accumulation in Arabidopsis

Nitric oxide (NO) generation by NO synthase (NOS) in guard cells plays a vital role in stomatal closure for adaptive plant response to drought stress. However, the mechanism underlying the regulation of NOS activity in plants is unclear. Here, by screening yeast deletion mutants with decreased NO accumulation and NOS‐like activity when subjected to H₂O₂ stress, we identified TUP1 as a novel regulator of NOS‐like activity in yeast. Arabidopsis WD40‐REPEAT 5a (WDR5a), a homolog of yeast TUP1, complemented H₂O₂‐induced NO accumulation of a yeast mutant Δtup1, suggesting the conserved role of WDR5a in regulating NO accumulation and NOS‐like activity. This note was further confirmed by using an Arabidopsis RNAi line wdr5a‐1 and two T‐DNA insertion mutants of WDR5a with reduced WDR5a expression, in which both H₂O₂‐induced NO accumulation and stomatal closure were repressed. This was because H₂O₂‐induced NOS‐like activity was inhibited in the mutants compared with that of the wild type. Furthermore, these wdr5a mutants were more sensitive to drought stress as they had reduced stomatal closure and decreased expression of drought‐related genes. Together, our results revealed that WDR5a functions as a novel factor to modulate NOS‐like activity for changes of NO accumulation and stomatal closure in drought stress tolerance.     

Tandem crystallization strategies for resolution of 3,3,3‐trifluorolactic acid [CF3CH(OH)COOH] by chiral benzylamines

Resolution of rac‐3,3,3‐trifluorolactic acid by diastereomeric salt formation was reinvestigated. The use of (S)‐1‐phenylethylamine gives coprecipitation of two diastereomeric phases, 1 (S)‐[NH₃CH(CH₃)Ph](S)‐[CF₃CH(OH)COO] and 2 (S)‐[NH₃CH(CH₃)Ph](R)‐[CF₃CH(OH)COO]·H₂O. Pure phase 1 may be obtained using molecular sieves as desiccants. Resolution by (S,S)‐2‐amino‐1‐phenylpropan‐1,3‐diol gives monoclinic (S,S)‐[NH₃CH(CH₂OH)CHOHPh] (R)‐[CF₃CH(OH)‐COO] 3 with minor (S)‐3,3,3‐trifluorolactate contamination, which is precluded in the recrystallized orthorhombic form 4 . A new resolution using inexpensive phenylglycinol gives pure phase 5 (S)‐[NH₃CH(CH₂OH)Ph] (S)‐[CF₃CH(OH)COO] in 76% yield, 94% ee in a single step, in preference to its (S)‐(R) diastereomer 6 . Overall efficient resolution for both enantiomers of the trifluorolactic acid (each ca. 70% yield, 99% ee) may be achieved by various two‐step “tandem” crystallizations, involving direct addition of either water or a second base to the filtrate from the initial reaction.     

Deciphering the mechanisms involved in Portulaca oleracea (C4) response to drought: metabolic changes including crassulacean acid‐like metabolism induction and reversal upon re‐watering

Portulaca oleracea is a C₄ plant; however, under drought it can change its carbon fixation metabolism into a crassulacean acid metabolism (CAM)‐like one. While the C₃‐CAM shift is well known, the C₄‐CAM transition has only been described in Portulaca. Here, a CAM‐like metabolism was induced in P. oleracea by drought and then reversed by re‐watering. Physiological and biochemical approaches were undertaken to evaluate the drought and recovery responses. In CAM‐like plants, chlorophyll fluorescence parameters were transitory affected and non‐radiative energy dissipation mechanisms were induced. Induction of flavonoids, betalains and antioxidant machinery may be involved in photosynthetic machinery protection. Metabolic analysis highlights a clear metabolic shift, when a CAM‐like metabolism is induced and then reversed. Increases in nitrogenous compounds like free amino acids and urea, and of pinitol could contribute to withstand drought. Reciprocal variations in arginase and urease in drought‐stressed and in re‐watered plants suggest urea synthesis is strictly regulated. Recovery of C₄ metabolism was accounted by CO₂ assimilation pattern and malate levels. Increases in glycerol and in polyamines would be of importance of re‐watered plants. Collectively, in P. oleracea multiple strategies, from induction of several metabolites to the transitory development of a CAM‐like metabolism, participate to enhance its adaptation to drought.     

HCN4 knockdown in dorsal hippocampus promotes anxiety‐like behavior in mice

Hyperpolarization‐activated and cyclic nucleotide‐gated (HCN) channels mediate the I_h current in the murine hippocampus. Disruption of the I_h current by knockout of HCN1, HCN2 or tetratricopeptide repeat‐containing Rab8b‐interacting protein has been shown to affect physiological processes such as synaptic integration and maintenance of resting membrane potentials as well as several behaviors in mice, including depressive‐like and anxiety‐like behaviors. However, the potential involvement of the HCN4 isoform in these processes is unknown. Here, we assessed the contribution of the HCN4 isoform to neuronal processing and hippocampus‐based behaviors in mice. We show that HCN4 is expressed in various regions of the hippocampus, with distinct expression patterns that partially overlapped with other HCN isoforms. For behavioral analysis, we specifically modulated HCN4 expression by injecting recombinant adeno‐associated viral (rAAV) vectors mediating expression of short hairpin RNA against hcn4 (shHcn4) into the dorsal hippocampus of mice. HCN4 knockdown produced no effect on contextual fear conditioning or spatial memory. However, a pronounced anxiogenic effect was evident in mice treated with shHcn4 compared to control littermates. Our findings suggest that HCN4 specifically contributes to anxiety‐like behaviors in mice.     

Structure of the Y. pseudotuberculosis adhesin InvasinE

Enteropathogenic Yersinia expresses several invasins that are fundamental virulence factors required for adherence and colonization of tissues in the host. Within the invasin‐family of Yersinia adhesins, to date only Invasin has been extensively studied at both structural and functional levels. In this work, we structurally characterize the recently identified inverse autotransporter InvasinE from Yersinia pseudotuberculosis (formerly InvasinD from Yersinia pseudotuberculosis strain IP31758) that belongs to the invasin‐family of proteins. The sequence of the C‐terminal adhesion domain of InvasinE differs significantly from that of other members of the Yersinia invasin‐family and its detailed cellular and molecular function remains elusive. In this work, we present the 1.7 Å crystal structure of the adhesion domain of InvasinE along with two Immunoglobulin‐like domains. The structure reveals a rod shaped architecture, confirmed by small angle X‐ray scattering in solution. The adhesion domain exhibits strong structural similarities to the C‐type lectin‐like domain of Yersinia pseudotuberculosis Invasin and enteropathogenic/enterohemorrhagic E. coli Intimin. However, despite the overall structural similarity, the C‐type lectin‐like domain in InvasinE lacks motifs required for Ca²⁺/carbohydrate binding as well as sequence or structural features critical for Tir binding in Intimin and β₁‐integrin binding in Invasin, suggesting that InvasinE targets a distinct, yet unidentified molecule on the host‐cell surface. Although the biological role and target molecule of InvasinE remain to be elucidated, our structural data provide novel insights into the architecture of invasin‐family proteins and a platform for further studies towards unraveling the function of InvasinE in the context of infection and host colonization.