NSE5 subunit interacts with distant regions of the SMC arms in the Physcomitrium patens SMC5/6 complex.

Structural maintenance of chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P. patens Ppnse5KO1 mutant line with an N-terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5-PpSMC6 interaction in SMC5/6 loading to DNA lesions.

Structural insights into Rad18 targeting by the SLF1 BRCT domains.

Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair. The mechanism of the specific Rad18 recognition by SLF1 is unclear. Here, we present the crystal structure of the tandem BRCT repeat (tBRCT) in SLF1 (SLF1tBRCT) bound with the interacting Rad18 peptide. Our structure and biochemical studies demonstrate that SLF1tBRCT interacts with two phosphoserines and adjacent residues in Rad18 for high-affinity and specificity Rad18 recognition. We found that SLF1tBRCT utilizes mechanisms common among tBRCTs as well as unique ones for Rad18 binding, the latter include interactions with an α-helical structure in Rad18 that has not been observed in other tBRCT-bound ligand proteins. Our work provides structural insights into Rad18 targeting by SLF1 and expands the understanding of BRCT-mediated complex assembly.

Characterization of the conserved features of the NSE6 subunit of the Physcomitrium patens SMC5/6 complex.

Structural maintenance of chromosomes (SMC) complexes are molecular machines ensuring chromatin organization at higher levels. They play direct roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of long-armed SMC, kleisin, and kleisin-associated subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. In the human HsNSE6/SLF2, we recently identified a new CANIN domain. Here we tracked down its sequence homology to lower plants, selected the bryophyte Physcomitrium patens, and analyzed PpNSE6 protein-protein interactions to explore its conservation in detail. We identified a previously unrecognized core sequence motif conserved from yeasts to humans within the NSE6 CANIN domain. This motif mediates the interaction between NSE6 and its NSE5 partner in yeasts and plants. In addition, the CANIN domain and its preceding PpNSE6 sequences bind both PpSMC5 and PpSMC6 arms. Interestingly, we mapped the PpNSE6-binding site at the PpSMC5 arm right next to the PpNSE2-binding surface. The position of NSE6 at SMC arms suggests its role in the regulation of SMC5/6 dynamics. Consistent with the regulatory role of NSE6 subunits, Ppnse6 mutant lines were viable and sensitive to the DNA-damaging drug bleomycin and lost a large portion of rDNA copies. These moss mutants also exhibited reduced growth and developmental aberrations. Altogether, our data showed the conserved function of the NSE6 subunit and architecture of the SMC5/6 complex across species.

SLF2 Interacts with the SMC5/6 Complex to Direct Hepatitis B Virus Episomal DNA to Promyelocytic Leukemia Bodies for Transcriptional Repression.

Hepatitis B virus (HBV) chronically infects approximately 300 million people worldwide, and permanently repressing transcription of covalently closed circular DNA (cccDNA), the episomal viral DNA reservoir, is an attractive approach toward curing HBV. However, the mechanism underlying cccDNA transcription is only partially understood. In this study, by illuminating cccDNA of wild-type HBV (HBV-WT) and transcriptionally inactive HBV that bears a deficient HBV X gene (HBV-ΔX), we found that the HBV-ΔX cccDNA more frequently colocalizes with promyelocytic leukemia (PML) bodies than that of HBV-WT cccDNA. A small interfering RNA (siRNA) screen targeting 91 PML body-related proteins identified SMC5-SMC6 localization factor 2 (SLF2) as a host restriction factor of cccDNA transcription, and subsequent studies showed that SLF2 mediates HBV cccDNA entrapment in PML bodies by interacting with the SMC5/6 complex. We further showed that the region of SLF2 comprising residues 590 to 710 interacts with and recruits the SMC5/6 complex to PML bodies, and the C-terminal domain of SLF2 containing this region is necessary for repression of cccDNA transcription. Our findings shed new light on cellular mechanisms that inhibit HBV infection and lend further support for targeting the HBx pathway to repress HBV activity. IMPORTANCE Chronic HBV infection remains a major public health problem worldwide. Current antiviral treatments rarely cure the infection, as they cannot clear the viral reservoir, cccDNA, in the nucleus. Therefore, permanently silencing HBV cccDNA transcription represents a promising approach for a cure of HBV infection. Our study provides new insights into the cellular mechanisms that restrict HBV infection, revealing the role of SLF2 in directing HBV cccDNA to PML bodies for transcriptional repression. These findings have important implications for the development of antiviral therapies against HBV.

An S-locus F-box protein as pollen S determinant targets non-self S-RNase underlying self-incompatibility in Citrus.

Self-incompatibility (SI) is a crucial mechanism that prevents self-fertilization and inbreeding in flowering plants. Citrus exhibits SI regulated by a polymorphic S-locus containing an S-RNase gene and multiple S-locus F-box (SLF) genes. It has been documented that S-RNase functions as the pistil S determinant, but there is no direct evidence that the SLF genes closely linked with S-RNase function as pollen S determinants in Citrus. This study assembled the genomes of two pummelo (Citrus grandis) plants, obtained three novel complete and well-annotated S-haplotypes, and isolated 36 SLF or SLF-like alleles on the S-loci. Phylogenetic analysis of 138 SLFs revealed that the SLF genes were classified into 12 types, including six types with divergent or missing alleles. Furthermore, transformation experiments verified that the conserved S6-SLF7a protein can lead to the transition of SI to self-compatibility by recognizing non-self S8-RNase in 'Mini-Citrus' plants (S7S8 and S8S29, Fortunella hindsii), a model plant for citrus gene function studies. In vitro assays demonstrated interactions between SLFs of different S haplotypes and the Skp1-Cullin1-F-box subunit CgSSK1 protein. This study provides direct evidence that SLF controls the pollen function in Citrus, demonstrating its role in the 'non-self recognition' SI system.

Heat stress-induced NO enhanced perylenequinone biosynthesis of Shiraia sp. via calcium signaling pathway.

Perylenequinones (PQs) are natural photosensitizing compounds used as photodynamic therapy, and heat stress (HS) is the main limiting factor of mycelial growth and secondary metabolism of fungi. This study aimed to unravel the impact of HS-induced Ca2+ and the calcium signaling pathway on PQ biosynthesis of Shiraia sp. Slf14(w). Meanwhile, the intricate interplay between HS-induced NO and Ca2+ and the calcium signaling pathway was investigated. The outcomes disclosed that Ca2+ and the calcium signaling pathway activated by HS could effectively enhance the production of PQs in Shiraia sp. Slf14(w). Further investigations elucidated the specific mechanism through which NO signaling molecules induced by HS act upon the Ca2+/CaM (calmodulin) signaling pathway, thus propelling PQ biosynthesis in Shiraia sp. Slf14(w). This was substantiated by decoding the downstream positioning of the CaM/CaN (calcineurin) pathway in relation to NO through comprehensive analyses encompassing transcript levels, enzyme assays, and the introduction of chemical agents. Concurrently, the engagement of Ca2+ and the calcium signaling pathway in heat shock signaling was also evidenced. The implications of our study underscore the pivotal role of HS-induced Ca2+ and the calcium signaling pathway, which not only participate in heat shock signal transduction but also play an instrumental role in promoting PQ biosynthesis. Consequently, our study not only enriches our comprehension of the mechanisms driving HS signaling transduction in fungi but also offers novel insights into the PQ synthesis paradigm within Shiraia sp. Slf14(w). KEY POINTS: • The calcium signaling pathway was proposed to participate in PQ biosynthesis under HS. • HS-induced NO was revealed to act upon the calcium signaling pathway for the first time.

Heat stress enhanced perylenequinones biosynthesis of Shiraia sp. Slf14(w) through nitric oxide formation.

Perylenequinones (PQs) are a class of natural polyketides used as photodynamic therapeutics. Heat stress (HS) is an important environmental factor affecting secondary metabolism of fungi. This study investigated the effects of HS treatment on PQs biosynthesis of Shiraia sp. Slf14(w) and the underlying molecular mechanism. After the optimization of HS treatment conditions, the total PQs amount reached 577 ± 34.56 mg/L, which was 20.89-fold improvement over the control. Also, HS treatment stimulated the formation of intracellular nitric oxide (NO). Genome-wide analysis of Shiraia sp. Slf14(w) revealed iNOSL and cNOSL encoding inducible and constitutive NOS-like proteins (iNOSL and cNOSL), respectively. Cloned iNOSL in Escherichia coli BL21 showed higher nitric oxide synthase (NOS) activity than cNOSL, and the expression level of iNOSL under HS treatment was observably higher than that of cNOSL, suggesting that iNOSL is more responsible for NO production in the HS-treated strain Slf14(w) and may play an important role in regulating PQs biosynthesis. Moreover, the putative biosynthetic gene clusters for PQs and genes encoding iNOSL and nitrate reductase (NR) in the HS-treated strain Slf14(w) were obviously upregulated. PQs biosynthesis and efflux stimulated by HS treatment were significantly inhibited upon the addition of NO scavenger, NOS inhibitor, and NR inhibitor, indicating that HS-induced NO, as a signaling molecule, triggered promoted PQs biosynthesis and efflux. Our results provide an effective strategy for PQs production and contribute to the understanding of heat shock signal transduction studies of other fungi.Key points• PQs titer of Shiraia sp. Slf14(w) was significantly enhanced by HS treatment.• HS-induced NO was first reported to participate in PQs biosynthetic regulation.• Novel inducible and constitutive NOS-like proteins (iNOSL and cNOSL) were obtained and their NOS activities were determined.

White matter tract strength correlates with therapy outcome in persistent developmental stuttering.

Persistent stuttering is a prevalent neurodevelopmental speech disorder, which presents with involuntary speech blocks, sound and syllable repetitions, and sound prolongations. Affected individuals often struggle with negative feelings, elevated anxiety, and low self-esteem. Neuroimaging studies frequently link persistent stuttering with cortical alterations and dysfunctional cortico-basal ganglia-thalamocortical loops; dMRI data also point toward connectivity changes of the superior longitudinal fasciculus (SLF) and the frontal aslant tract (FAT). Both tracts are involved in speech and language functions, and the FAT also supports inhibitory control and conflict monitoring. Whether the two tracts are involved in therapy-associated improvements and how they relate to therapeutic outcomes is currently unknown. Here, we analyzed dMRI data of 22 patients who participated in a fluency-shaping program, 18 patients not participating in therapy, and 27 fluent control participants, measured 1 year apart. We used diffusion tractography to segment the SLF and FAT bilaterally and to quantify their microstructural properties before and after a fluency-shaping program. Participants learned to speak with soft articulation, pitch, and voicing during a 2-week on-site boot camp and computer-assisted biofeedback-based daily training for 1 year. Therapy had no impact on the microstructural properties of the two tracts. Yet, after therapy, stuttering severity correlated positively with left SLF fractional anisotropy, whereas relief from the social-emotional burden to stutter correlated negatively with right FAT fractional anisotropy. Thus, posttreatment, speech motor performance relates to the left dorsal stream, while the experience of the adverse impact of stuttering relates to the structure recently associated with conflict monitoring and action inhibition.

L-Arginine enhanced perylenequinone production in the endophytic fungus Shiraia sp. Slf14(w) via NO signaling pathway.

Perylenequinones (PQ) are natural polyketides used as anti-microbial, -cancers, and -viral photodynamic therapy agents. Herein, the effects of L-arginine (Arg) on PQ biosynthesis of Shiraia sp. Slf14(w) and the underlying molecular mechanism were investigated. The total content of PQ reached 817.64 ± 72.53 mg/L under optimal conditions of Arg addition, indicating a 30.52-fold improvement over controls. Comparative transcriptome analysis demonstrated that Arg supplement promoted PQ precursors biosynthesis of Slf14(w) by upregulating the expression of critical genes associated with the glycolysis pathway, and acetyl-CoA and malonyl-CoA synthesis. By downregulating the expression of genes related to the glyoxylate cycle pathway and succinate dehydrogenase, more acetyl-CoA flow into the formation of PQ. Arg supplement upregulated the putative biosynthetic gene clusters for PQ and activated the transporter proteins (MFS and ABC) for exudation of PQ. Further studies showed that Arg increased the gene transcription levels of nitric oxide synthase (NOS) and nitrate reductase (NR), and activated NOS and NR, thus promoting the formation of nitric oxide (NO). A supplement of NO donor sodium nitroprusside (SNP) also confirmed that NO triggered promoted biosynthesis and efflux of PQ. PQ production stimulated by Arg or/and SNP can be significantly inhibited upon the addition of NO scavenger carboxy-PTIO, NOS inhibitor Nω-nitro-L-arginine, or soluble guanylate cyclase inhibitor NS-2028. These results showed that Arg-derived NO, as a signaling molecule, is involved in the biosynthesis and regulation of PQ in Slf14(W) through the NO-cGMP-PKG signaling pathway. Our results provide a valuable strategy for large-scale PQ production and contribute to further understanding of NO signaling in the fungal metabolite biosynthesis. KEY POINTS: • PQ production of Shiraia sp. Slf14(w) was significantly improved by L-arginine addition. • Arginine-derived NO was firstly reported to be involved in the biosynthesis and regulation of PQ. • The NO-cGMP-PKG signaling pathway was proposed for the first time to participate in PQ biosynthesis.

Primary restriction of S-RNase cytotoxicity by a stepwise ubiquitination and degradation pathway in Petunia hybrida.

In self-incompatible Petunia species, the pistil S-RNase acts as cytotoxin to inhibit self-pollination but is polyubiquitinated by the pollen-specific nonself S-locus F-box (SLF) proteins and subsequently degraded by the ubiquitin-proteasome system (UPS), allowing cross-pollination. However, it remains unclear how S-RNase is restricted by the UPS. Using biochemical analyses, we first show that Petunia hybrida S3 -RNase is largely ubiquitinated by K48-linked polyubiquitin chains at three regions, R I, R II and R III. R I is ubiquitinated in unpollinated, self-pollinated and cross-pollinated pistils, indicating its occurrence before PhS3 -RNase uptake into pollen tubes, whereas R II and R III are exclusively ubiquitinated in cross-pollinated pistils. Transgenic analyses showed that removal of R II ubiquitination resulted in significantly reduced seed sets from cross-pollination and that of R I and R III to a lesser extent, indicating their increased cytotoxicity. Consistent with this, the mutated R II of PhS3 -RNase resulted in a marked reduction of its degradation, whereas that of R I and R III resulted in less reduction. Taken together, we demonstrate that PhS3 -RNase R II functions as a major ubiquitination region for its destruction and R I and R III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross-pollination in P. hybrida.

Investigating the effects of the core nitrogen atom configuration on the thermodynamic solubility of 6,5-bicyclic heterocycles.

Physicochemical properties, such as solubility, are important when prioritising compounds for progression on a drug discovery project. There is limited literature around the systematic effects of core changes on thermodynamic solubility. This work details the synthesis of nitrogen containing 6,5-bicyclic heterocyclic cores which are common scaffolds in medicinal chemistry and the analysis of their physicochemical properties, particularly, thermodynamic solubility. Crystalline solids were obtained where possible to enable a robust comparison of the thermodynamic solubility. Other parameters such as pKa, melting point and lipophilicity were also measured to determine the key factors affecting the observed solubility.

Methods and Experiments for Sensing Variations in Solar Activity and Defining Their Impact on Heart Variability.

This paper evaluates variations in solar activity and their impact on the human nervous system, including the manner in which human behavior and decision-making reflect such effects in the context of (symmetrical) social interactions. The relevant research showed that solar activity, manifesting itself through the exposure of the Earth to charged particles from the Sun, affects heart variability. The evaluation methods focused on examining the relationships between selected psychophysiological data and solar activity, which generally causes major alterations in the low-level electromagnetic field. The investigation within this paper revealed that low-level EMF changes are among the factors affecting heart rate variability and, thus, also variations at the spectral level of the rate, in the VLF, (f = 0.01-0.04 Hz), LF (f = 0.04-0.15 Hz), and HF (f = 0.15 az 0.40 Hz) bands. The results of the presented experiments can also be interpreted as an indirect explanation of sudden deaths and heart failures.

Altered White Matter Organization in the TUBB3 E410K Syndrome.

Seven unrelated individuals (four pediatric, three adults) with the TUBB3 E410K syndrome, harboring identical de novo heterozygous TUBB3 c.1228 G>A mutations, underwent neuropsychological testing and neuroimaging. Despite the absence of cortical malformations, they have intellectual and social disabilities. To search for potential etiologies for these deficits, we compared their brain's structural and white matter organization to 22 controls using structural and diffusion magnetic resonance imaging. Diffusion images were processed to calculate fractional anisotropy (FA) and perform tract reconstructions. Cortical parcellation-based network analysis and gyral topology-based FA analyses were performed. Major interhemispheric, projection and intrahemispheric tracts were manually segmented. Subjects had decreased corpus callosum volume and decreased network efficiency. While only pediatric subjects had diffuse decreases in FA predominantly affecting mid- and long-range tracts, only adult subjects had white matter volume loss associated with decreased cortical surface area. All subjects showed aberrant corticospinal tract trajectory and bilateral absence of the dorsal language network long segment. Furthermore, pediatric subjects had more tracts with decreased FA compared with controls than did adult subjects. These findings define a TUBB3 E410K neuroimaging endophenotype and lead to the hypothesis that the age-related changes are due to microscopic intrahemispheric misguided axons that are pruned during maturation.

Tractography and the connectome in neurosurgical treatment of gliomas: the premise, the progress, and the potential.

The ability of diffusion tensor MRI to detect the preferential diffusion of water in cerebral white matter tracts enables neurosurgeons to noninvasively visualize the relationship of lesions to functional neural pathways. Although viewed as a research tool in its infancy, diffusion tractography has evolved into a neurosurgical tool with applications in glioma surgery that are enhanced by evolutions in crossing fiber visualization, edema correction, and automated tract identification. In this paper the current literature supporting the use of tractography in brain tumor surgery is summarized, highlighting important clinical studies on the application of diffusion tensor imaging (DTI) for preoperative planning of glioma resection, and risk assessment to analyze postoperative outcomes. The key methods of tractography in current practice and crucial white matter fiber bundles are summarized. After a review of the physical basis of DTI and post-DTI tractography, the authors discuss the methodologies with which to adapt DT image processing for surgical planning, as well as the potential of connectomic imaging to facilitate a network approach to oncofunctional optimization in glioma surgery.

13 C NMR investigations of molecular order of rod-like, bent-core, and thiophene mesogens.

In this review, methods to obtain the orientational order of topologically variant molecular mesogens using by one- and two-dimensional (2D) solid-state 13 C nuclear magnetic resonance (NMR) spectroscopy are described. Besides 13 C chemical shifts, the 13 C─1 H dipolar couplings measured from 2D-separated local field (SLF) technique are used for computing the order parameters of a variety of mesogens. The investigated molecules are composed of a variable number of rings in the core, that is, core ranging from simply one ring to five rings. Among the mesogens investigated, a special focus has been placed on mesogens with thiophene rings, which are gaining popularity as liquid crystalline organic semiconductors. The replacement of a phenyl ring by thiophene in the core has a dramatic influence on molecular topology, as observed from the measured order parameters. The review highlights the advantages of the 2D SLF method for understanding the local dynamics and for mapping the topology of mesogens through the measured order parameters. SLF NMR studies of as many as 24 molecular mesogens that vary in terms of the molecular structure as well as topology are covered in the review. Order parameters of the rings have been estimated from the 13 C─1 H dipolar couplings in the nematic, smectic A, smectic C, and tilted hexatic phases as well as in B1 and B2 mesophases of various mesogens. It is anticipated that, in the years to come, the 2D SLF method would provide advanced molecular information on structurally complex mesogens that are emerging in liquid crystal science through the incessant efforts of synthetic chemists. The mini review covers the orientational order of topologically variant molecular mesogens determined by 1D and 2D solid-state 13 C NMR spectroscopy. Accordingly, rod-like, bent-core, and thiophene mesogens were subjected to 2D SLF measurements to get the order parameters from which the topology was established. The replacement of phenyl ring by thiophene and its influence on order parameters as well as on molecular topology is also discussed.

When imaging meets neurophysiology: the value of navigated transcranial magnetic stimulation for preoperative neurophysiological mapping prior to brain tumor surgery.

Maximal safe resection is the modern goal for surgery of intrinsic brain tumors located in or close to brain eloquent areas. Nowadays different neuroimaging techniques provide important anatomical and functional information regarding the brain functional organization that can be used to plan a customized surgical strategy to preserve functional networks, and to increase the extent of tumor resection. Among these techniques, navigated transcranial magnetic stimulation (nTMS) has recently gained great favor among the neurosurgical community for preoperative mapping and planning prior to brain tumor surgery. It represents an advanced neuroimaging technique based on the neurophysiological mapping of the functional cortical brain organization. Moreover, it can be combined with other neuroimaging techniques such as diffusion tensor imaging tractography, thus providing a reliable reconstruction of brain eloquent networks. Consequently, nTMS mapping may provide reliable noninvasive brain functional mapping, anticipating information that otherwise may be available to neurosurgeons only in the operating theater by using direct electrical stimulation. The authors describe the reliability and usefulness of the preoperative nTMS-based approach in neurosurgical practice, and briefly discuss their experience using nTMS as well as currently available evidence in the literature supporting its clinical use. In particular, special attention is reserved for the discussion of the role of nTMS as a novel tool for the preoperative neurophysiological mapping of motor and language networks prior to surgery of intrinsic brain tumors located in or close to eloquent networks, as well as for future and promising applications of nTMS in neurosurgical practice.

Electrostatic potentials of the S-locus F-box proteins contribute to the pollen S specificity in self-incompatibility in Petunia hybrida.

Self-incompatibility (SI) is a self/non-self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S-locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S-locus encodes a single S-RNase and a cluster of S-locus F-box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of 'like charges repel and unlike charges attract' between SLFs and S-RNases in Petunia hybrida. Strikingly, the alteration of a single C-terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S-RNases, providing a mechanistic insight into the self/non-self discrimination between cytosolic proteins in angiosperms.

Enhanced production of perylenequinones in the endophytic fungus Shiraia sp. Slf14 by calcium/calmodulin signal transduction.

Perylenequinones (PQ) that notably produce reactive oxygen species upon exposure to visible light are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens such as Shiraia sp. The involvement of Ca2+/calmodulin (CaM) signalling in PQ biosynthesis was investigated by submerged culturing of Shiraia sp. Slf14, a species that produces hypocrellins HA and HB and elsinochromes EA, EB, and EC. Our results showed that the total content of PQ reached 1894.66 ± 21.93 mg/L under optimal conditions of Ca2+ addition, which represents a 5.8-fold improvement over controls. The addition of pharmacological Ca2+ sensor inhibitors strongly inhibited PQ production, which indicates that Ca2+/CaM signalling regulates PQ biosynthesis. The expression levels of Ca2+ sensor and PQ biosynthetic genes were downregulated following addition of inhibitors but were upregulated upon addition of Ca2+. Inhibition was partially released by external Ca2+ supplementation. Fluo-3/AM experiments revealed that similar cytosolic Ca2+ variation occurred under these conditions. These results demonstrated that Ca2+ signalling via the CaM transduction pathway plays a pivotal role in PQ biosynthesis.

Design and development of a biorelevant simulated human lung fluid.

Biorelevant fluids are required to enable meaningful in vitro experimental determinations of the biopharmaceutical properties of inhaled medicines, e.g. drug solubility, particle dissolution, cellular uptake. Our aim was to develop a biorelevant simulated lung fluid (SLF) with a well-defined composition and evidence-based directions for use. The SLF contained dipalmitoylphosphotidylcholine, dipalmitoylphosphatidylglycerol, cholesterol, albumin, IgG, transferrin and antioxidants. Freshly made SLF had pH 7.2, viscosity 1.138 × 10-3 Pa s, conductivity 14.5 mS/m, surface tension 54.9 mN/m and density 0.999 g/cm3. Colour, surface tension and conductivity were the most sensitive indicators of product deterioration. The simulant was stable for 24 h and 48 h at 37 °C and 21 °C, respectively, (in-use stability) and for 14 days when stored in a refrigerator (storage stability). To extend stability, the SLF was vacuum freeze-dried in batches to produce lyophilised powder that can be reconstituted readily when needed at the point of use. In conclusion, we have reported the composition and manufacture of a biorelevant, synthetic SLF, provided a detailed physico-chemical characterisation and recommendations for how to store and use a product that can be used to generate experimental data to provide inputs to computational models that predict drug bioavailability in the lungs.

Olive oil bioactive compounds increase body weight, and improve gut health and integrity in gilthead sea bream (Sparus aurata).

An olive oil bioactive extract (OBE) rich in bioactive compounds like polyphenols, triterpenic acids, long-chain fatty alcohols, unsaturated hydrocarbons, tocopherols and sterols was tested (0, 0·08, 0·17, 0·42 and 0·73 % OBE) in diets fed to sea bream (Sparus aurata) (initial weight: 5·4 (sd 1·2) g) during a 90-d trial (four replicates). Fish fed diets containing 0·17 and 0·42 % OBE were 5 % heavier (61·1 (sd 1·6) and 60·3 (sd 1·1) g, respectively) than those of the control group (57·0 (sd 0·7) g), although feed conversion ratio and specific feed intake did not vary. There were no differences in lipid peroxidation (LPO) levels, catalase, glutathione reductase and glutathione S-transferase activities in the intestine and liver, although there was a tendency of lower intestinal and hepatic LPO levels in fish fed OBE diets. No differences in villus size were found among treatments, whereas goblet cell density in the control group was on average14·3 % lower than in fish fed OBE diets. The transcriptomic profiling of intestinal markers, covering different biological functions like (i) cell differentiation and proliferation, (ii) intestinal permeability, (iii) enterocyte mass and epithelial damage, (iv) IL and cytokines, (v) pathogen recognition receptors and (vi) mitochondria function, indicated that among the eighty-eight evaluated genes, twenty-nine were differentially expressed (0·17 % OBE diet), suggesting that the additive has the potential of improving the condition and defensive role of the intestine by enhancing the maturation of enterocytes, reducing oxidative stress, improving the integrity of the intestinal epithelium and enhancing the intestinal innate immune function, as gene expression data indicated.