Cloning and characterization of four enzymes responsible for cyclohexylamine degradation from Paenarthrobacter sp. TYUT067.

Paenarthrobacter sp. TYUT067 is a soil bacterium that can degrade and use cyclohexylamine as the sole source of carbon and energy. However, the responsible enzymes involved in cyclohexylamine degradation by TYUT067 have not been cloned and characterized in detail yet. In this study, four possible cyclohexylamine degradation genes, one cyclohexylamine oxidase (Pachao), two cyclohexanone monooxygenases (Pachms) and one lactone hydrolase (Pamlh) were successfully cloned and heterologous expressed in Escherichia coli T7 host cells. The four enzymes were purified and characterized. The optimal pH and temperature of the purified enzymes toward their own substrates were 7.0 (PaCHAO), 8.0 (PaCHM1), 9.0 (PaCHM2 and PaMLH) and 30 °C (PaCHAO and PaMLH), 40 °C (PaCHM2) and 45 °C (PaCHM1), respectively, with KM of 1.1 mM (PaCHAO), 0.1 mM (PaCHM1), 0.1 mM (PaCHM2) and 0.8 mM (PaMLH), and yielding a catalytic efficiency kcat/KM of 16.1 mM-1 s-1 (PaCHAO), 1.0 mM-1 s-1 (PaCHM1), 5.0 mM-1 s-1 (PaCHM2) and 124.4 mM-1 s-1 (PaMLH). In vitro mimicking the cyclohexylamine degradation pathway was conducted by using the combined three cyclohexylamine degradation enzymes (PaCHAO, PaCHM2 and PaMLH) with 10-50 mM cyclohexylamine, 100% conversion of cyclohexylamine could be finished within 12 h without any detected intermediates. The current study confirmed the enzymes responsible for cyclohexylamine degradation in TYUT067 for the first time, provide basic information for further investigation and application of these specific enzymes in pollution control.

The soluble epoxide hydrolase inhibitor GSK2256294 decreases the proportion of adipose pro-inflammatory T cells.

Adipose tissue contains a complex immune environment and is a central contributor to heightened systemic inflammation in obese persons. Epoxyeicosatrienoic acids (EETs) are lipid signaling molecules that decrease inflammation in obese animals, but their effect on inflammation in humans is unknown. The enzyme soluble epoxide hydrolase (sEH) hydrolyzes EETs to less active diols, and we hypothesized that pharmacologic sEH inhibition would decrease adipose inflammation in obese individuals. We treated obese prediabetic adults with the sEH inhibitor GSK2256294 versus placebo in a crossover design, collected subcutaneous abdominal adipose tissue via lipoaspiration and characterized the tissue T cell profile. Treatment with GSK2256294 decreased the percentage of pro-inflammatory T cells producing interferon-gamma (IFNγ), but not interleukin (IL)-17A, and decreased the amount of secreted tumor necrosis factor-alpha (TNFα). Understanding the contribution of the EET/sEH pathway to inflammation in obesity could lead to new strategies to modulate adipose and systemic inflammation.

A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of hOAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1R,3S,4S)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (1), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying 8 and 9 as novel selective hOAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated 8 and 9 as covalent inhibitors of hOAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel addition-aromatization inactivation mechanism for 9 contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.

Targeting ferroptosis in rhabdomyosarcoma cells.

Recent data suggest that rhabdomyosarcoma (RMS) cells might be vulnerable to oxidative stress-induced cell death. Here, we show that RMS are susceptible to cell death induced by Erastin, an inhibitor of the glutamate/cystine antiporter xc- that can increase reactive oxygen species (ROS) production via glutathione (GSH) depletion. Prior to cell death, Erastin caused GSH depletion, ROS production and lipid peroxidation. Importantly, pharmacological inhibitors of lipid peroxidation (i.e., Ferrostatin-1, Liproxstatin-1), ROS scavengers (i.e., α-Tocopherol, GSH) and the iron chelator Deferoxamine inhibited ROS accumulation, lipid peroxidation and cell death, consistent with ferroptosis. Interestingly, the broad-spectrum protein kinase C (PKC) inhibitor Bisindolylmaleimide I as well as the PKCα- and ß-selective inhibitor Gö6976 significantly reduced Erastin-induced cell death. Similarly, genetic knockdown of PKCα significantly protected RMS cells from Erastin-induced cell death. Furthermore, the broad-spectrum nicotinamide adenine dinucleotide phosphate-oxidase (NOX) inhibitor Diphenyleneiodonium and the selective NOX1/4 isoform inhibitor GKT137831 significantly decreased Erastin-stimulated ROS, lipid ROS and cell death. These data provide new insights into the molecular mechanisms of ferroptosis in RMS, contributing to the development of new redox-based treatment strategies.

Hedgehog signaling stimulates the formation of proliferating Müller glia-derived progenitor cells in the chick retina.

Müller glia can be stimulated to de-differentiate and become proliferating progenitor cells that regenerate neurons in the retina. The signaling pathways that regulate the formation of proliferating Müller glia-derived progenitor cells (MGPCs) are beginning to be revealed. The purpose of this study was to investigate whether Hedgehog (Hh) signaling influences the formation of MGPCs in the chick retina. We find that Hh signaling is increased in damaged retinas where MGPCs are known to form. Sonic Hedgehog (Shh) is normally present in the axons of ganglion cells, but becomes associated with Müller glia and MGPCs following retinal damage. Activation of Hh signaling with recombinant human SHH (rhShh) or smoothened agonist (SAG) increased levels of Ptch1, Gli1, Gli2, Gli3, Hes1 and Hes5, and stimulated the formation of proliferating MGPCs in damaged retinas. In undamaged retinas, SAG or rhShh had no apparent effect upon the Müller glia. However, SAG combined with FGF2 potentiated the formation of MGPCs, whereas SAG combined with IGF1 stimulated the nuclear migration of Müller glia, but not the formation of MGPCs. Conversely, inhibition of Hh signaling with KAAD-cyclopamine, Gli antagonists or antibody to Shh reduced numbers of proliferating MGPCs in damaged and FGF2-treated retinas. Hh signaling potentiates Pax6, Klf4 and cFos expression in Müller glia during the formation of MGPCs. We find that FGF2/MAPK signaling recruits Hh signaling into the signaling network that drives the formation of proliferating MGPCs. Our findings implicate Hh signaling as a key component of the network of signaling pathways that promote the de-differentiation of Müller glia and proliferation of MGPCs.

Lipid Peroxidation-Dependent Cell Death Regulated by GPx4 and Ferroptosis.

Glutathione peroxidase 4 (Phospholipid hydroperoxide glutathione peroxidase, PHGPx) can directly reduce phospholipid hydroperoxide. Depletion of GPx4 induces lipid peroxidation-dependent cell death in embryo, testis, brain, liver, heart, and photoreceptor cells of mice. Administration of vitamin E in tissue specific GPx4 KO mice restored tissue damage in testis, liver, and heart. These results indicate that suppression of phospholipid peroxidation is essential for cell survival in normal tissues in mice. Ferroptosis is an iron-dependent non-apoptotic cell death that can elicited by pharmacological inhibiting the cystine/glutamate antiporter, system Xc- (type I) or directly binding and loss of activity of GPx4 (Type II) in cancer cells with high level RAS-RAF-MEK pathway activity or p53 expression, but not in normal cells. Ferroptosis by Erastin (Type I) and RSL3 (RAS-selective lethal 3, Type II) treatment was suppressed by an iron chelator, vitamin E and Ferrostatin-1, antioxidant compound. GPx4 can regulate ferroptosis by suppression of phospholipid peroxidation in erastin and RSL3-induced ferroptosis. Recent works have identified several regulatory factors of erastin and RSL3-induced ferroptosis. In our established GPx4-deficient MEF cells, depletion of GPx4 induce iron and 15LOX-independent lipid peroxidation at 26 h and caspase-independent cell death at 72 h, whereas erastin and RSL3 treatment resulted in iron-dependent ferroptosis by 12 h. These results indicated the possibility that the mechanism of GPx4-depleted cell death might be different from that of ferroptosis induced by erastin and RSL3.

t-BuOOH induces ferroptosis in human and murine cell lines.

Reactive oxygen species (ROS)-induced apoptosis has been extensively studied. Increasing evidence suggests that ROS, for instance, induced by hydrogen peroxide (H2O2), might also trigger regulated necrotic cell death pathways. Almost nothing is known about the cell death pathways triggered by tertiary-butyl hydroperoxide (t-BuOOH), a widely used inducer of oxidative stress. The lipid peroxidation products induced by t-BuOOH are involved in the pathophysiology of many diseases, such as cancer, cardiovascular diseases, or diabetes. In this study, we exposed murine fibroblasts (NIH3T3) or human keratinocytes (HaCaT) to t-BuOOH (50 or 200 µM, respectively) which induced a rapid necrotic cell death. Well-established regulators of cell death, i.e., p53, poly(ADP)ribose polymerase-1 (PARP-1), the stress kinases p38 and c-Jun N-terminal-kinases 1/2 (JNK1/2), or receptor-interacting serine/threonine protein kinase 1 (RIPK1) and 3 (RIPK3), were not required for t-BuOOH-mediated cell death. Using the selective inhibitors ferrostatin-1 (1 µM) and liproxstatin-1 (1 µM), we identified ferroptosis, a recently discovered cell death mechanism dependent on iron and lipid peroxidation, as the main cell death pathway. Accordingly, t-BuOOH exposure resulted in a ferrostatin-1- and liproxstatin-1-sensitive increase in lipid peroxidation and cytosolic ROS. Ferroptosis was executed independently from other t-BuOOH-mediated cellular damages, i.e., loss of mitochondrial membrane potential, DNA double-strand breaks, or replication block. H2O2 did not cause ferroptosis at equitoxic concentrations (300 µM) and induced a (1) lower and (2) ferrostatin-1- or liproxstatin-1-insensitive increase in lipid peroxidation. We identify that t-BuOOH and H2O2 produce a different pattern of lipid peroxidation, thereby leading to different cell death pathways and present t-BuOOH as a novel inducer of ferroptosis.

Draft Genome Sequence of Cyclohexylamine-Degrading Strain Acinetobacter sp. YT-02 Isolated.

Acinetobacter sp. YT-02, a Gram-negative bacterium isolated from the activated sludge from a sodium N-cyclohexylsulfamate production plant, has the ability to degrade cyclohexylamine. It was classified as a member of Acinetobacter sp., a Gram-negative bacterium, sharing a 16S rRNA gene sequence identity of 99% with Acinetobacter guangdongensis strain 1NM-4. It could degrade 10 mmol/L cyclohexylamine within 22 h. Based on the identified metabolite, the metabolic pathway of cyclohexylamine could be postulated as it was degraded via cyclohexanone. Draft genome sequence of this strain (2,993, 647 bp of chromosome length) is presented here. We further identified the genes encoding the enzymes involved in cyclohexylamine oxidation to cyclohexanone and the subsequent downstream metabolic pathway of cyclohexanone oxidation. Strain YT-02 has the potentiality to be applied in the treatment of the pollutant cyclohexylamine, and it could also be treated as a research material to study the degradation mechanism of cyclohexylamine.

Fluoro-Carba-Sugars are Glycomimetic Activators of the glmS Ribozyme.

The glmS ribozyme is a bacterial gene-regulating riboswitch that controls cell wall synthesis, depending on glucosamine-6-phosphate as a cofactor. Due to the presence of this ribozyme in several human pathogen bacteria (e.g., MRSA, VRSA), the glmS ribozyme represents an attractive target for the development of artificial cofactors. The substitution of the ring oxygen in carbohydrates by functionalized methylene groups leads to a new generation of glycomimetics that exploits distinct interaction possibilities with their target structure in biological systems. Herein, we describe the synthesis of mono-fluoro-modified carba variants of α-d-glucosamine and ß-l-idosamine. (5aR)-Fluoro-carba-α-d-glucosamine-6-phosphate is a synthetic mimic of the natural ligand of the glmS ribozyme and is capable of effectively addressing its unique self-cleavage mechanism. However, in contrast to what was expected, the activity is significantly decreased compared to its non-fluorinated analog. By combining self-cleavage assays with the Bacillus subtilis and Staphylococcus aureus glmS ribozyme and molecular docking studies, we provide a structure-activity relationship for fluorinated carba-sugars.

Comparative Profiling and Discovery of Novel Glycosylated Mycosporine-Like Amino Acids in Two Strains of the Cyanobacterium Scytonema cf. crispum.

UNLABELLED: The mycosporine-like amino acids (MAAs) are a group of small molecules with a diverse ecological distribution among microorganisms. MAAs have a range of physiological functions, including protection against UV radiation, making them important from a biotechnological perspective. In the present study, we identified a putative MAA (mys) gene cluster in two New Zealand isolates of Scytonema cf. crispum (UCFS10 and UCFS15). Homology to "Anabaena-type" mys clusters suggested that this cluster was likely to be involved in shinorine biosynthesis. Surprisingly, high-performance liquid chromatography analysis of S cf. crispum cell extracts revealed a complex MAA profile, including shinorine, palythine-serine, and their hexose-bound variants. It was hypothesized that a short-chain dehydrogenase (UCFS15_00405) encoded by a gene adjacent to the S cf. crispum mys cluster was responsible for the conversion of shinorine to palythine-serine. Heterologous expression of MysABCE and UCFS15_00405 in Escherichia coli resulted in the exclusive production of the parent compound shinorine. Taken together, these results suggest that shinorine biosynthesis in S cf. crispum proceeds via an Anabaena-type mechanism and that the genes responsible for the production of other MAA analogues, including palythine-serine and glycosylated analogues, may be located elsewhere in the genome. IMPORTANCE: Recently, New Zealand isolates of S cf. crispum were linked to the production of paralytic shellfish toxins for the first time, but no other natural products from this species have been reported. Thus, the species was screened for important natural product biosynthesis. The mycosporine-like amino acids (MAAs) are among the strongest absorbers of UV radiation produced in nature. The identification of novel MAAs is important from a biotechnology perspective, as these molecules are able to be utilized as sunscreens. This study has identified two novel MAAs that have provided several new avenues of future research related to MAA genetics and biosynthesis. Further, we have revealed that the genetic basis of MAA biosynthesis may not be clustered on the genome. The identification of the genes responsible for MAA biosynthesis is vital for future genetic engineering.

Novel bisnaphthalimidopropyl (BNIPs) derivatives as anticancer compounds targeting DNA in human breast cancer cells.

Bisnaphthalimidopropyl (BNIP) derivatives are a family of compounds that exert anti-cancer activities in vitro and, according to previous studies, variations in the linker sequence have increased their DNA binding and cytotoxic activities. By modifying the linker sequence of bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM), a previously synthesised BNIP derivative with anti-cancer properties, three novel BNIP derivatives were designed. Bisnaphthalimidopropyl-piperidylpropane (BNIPPiProp), a structural isomer of BNIPDaCHM, bisnaphthalimidopropyl ethylenedipiperidine dihydrobromide (BNIPPiEth), an isoform of BNIPDaCHM with a shorter linker chain, and (trans(trans))-bisnaphthalimidopropyl diaminodicyclohexylmethane (trans,trans-BNIPDaCHM), a stereoisomer of BNIPDaCHM, were successfully synthesised (72.3-29.5% yield) and characterised by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). Competitive displacement of ethidium bromide (EtBr) and UV binding studies were used to study the interactions of BNIP derivatives with Calf Thymus DNA. The cytotoxicity of these derivatives was assessed against human breast cancer MDA-MB-231 and SKBR-3 cells by MTT assay. Propidium iodide (PI) flow cytometry was conducted in order to evaluate the cellular DNA content in both breast cancer cell lines before and after treatment with BNIPs. The results showed that all novel BNIPs exhibit strong DNA binding properties in vitro, and strong cytotoxicity, with IC50 values in the range of 0.2-3.3 µM after 24 hours drug treatment. Two of the novel BNIP derivatives, BNIPPiEth and trans,trans-BNIPDaCHM, exhibited greater cytotoxicity against the two breast cancer cell lines studied, compared to BNIPDaCHM. By synthesising enantiopures and reducing the length of the linker sequence, the cytotoxicity of the BNIP derivatives was significantly improved compared to BNIPDaCHM, while maintaining DNA binding and bis-intercalating properties. In addition, cell cycle studies indicated that trans,trans-BNIPDaCHM, the most cytotoxic BNIP derivative, induced sub-G1 cell cycle arrest, indicative of apoptotic cell death. Based on these findings, further investigation is under way to assess the potential efficacy of trans,trans-BNIPDaCHM and BNIPPiEth in treating human breast cancer.

Salting-out-assisted liquid-liquid extraction as a suitable approach for determination of methoxetamine in large sets of tissue samples.

A new designer drug, a dissociative anesthetic, and a putative N-methyl-D-aspartate receptor antagonist, methoxetamine (MXE) noted by the EU Early Warning System has been already identified as a cause of several fatalities worldwide. The primary objective of this work was to develop a suitable sample preparation method allowing for isolation of MXE and its main metabolites in high yields from rat brain, liver, and lungs. For the purpose of the project, MXE and five metabolites were synthesized in-house, specifically O-desmethyl-normethoxetamine, O-desmethylmethoxetamine, dihydro-O-desmethylmethoxetamine, normethoxetamine, and dihydromethoxetamine. A sample preparation procedure consisted in the homogenization of the tissue applying salting-out-assisted liquid-liquid extraction (SALLE). A subsequent liquid chromatography-mass spectrometry (LC-MS) analysis was based on reversed-phased chromatography hyphenated with a triple quad MS system in a positive electrospray mode. Multiple reaction monitoring (MRM) was used for qualification and quantification of the analytes. The quantification was based on the application of an isotopically labeled internal standard, normethoxetamine-d3. The matrix-matched calibrations were prepared for each type of matrix with regression coefficients 0.9943-1.0000. The calibration curves were linear in the concentration range of 2.5-250 ng g(-1). Limits of quantification (LOQs) were estimated as 2.5 and 5 ng g(-1), respectively. Recovery (80-117%) and matrix effect (94-110%) at 100 ng g(-1) and intra- and inter-day accuracy and precision at low (2.5 ng g(-1)), middle (25 ng g(-1)), and upper (250 ng g(-1)) concentration levels for all the analytes in all three types of tissues were also determined. The developed analytical method was applied to a set of real samples gathered in toxicological trials on rats and MXE, and its metabolites were determined successfully.

Functional assessment of mycosporine-like amino acids in Microcystis aeruginosa strain PCC 7806.

The biological role of the widespread mycosporine-like amino acids (MAAs) in cyanobacteria is under debate. Here, we have constructed and characterized two mutants impaired in MAA biosynthesis in the bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806. We could identify shinorine as the sole MAA type of the strain, which is exclusively located in the extracellular matrix. Bioinformatic studies as wells as polymerase chain reaction screening revealed that the ability to produce MAAs is sporadically distributed within the genus. Growth experiments and reactive oxygen species quantification with wild-type and mutant strains did not support a role of shinorine in protection against UV or other stress conditions in M. aeruginosa PCC 7806. The shinorine content per dry weight of cells as well as transcription of the mys gene cluster was not significantly elevated in response to UV-A, UV-B or any other stress condition tested. Remarkably, both mutants exhibited pronounced morphological changes compared with the wild type. We observed an increased accumulation and an enhanced hydrophobicity of the extracellular matrix. Our study suggests that MAAs in Microcystis play a negligible role in protection against UV radiation but might be a strain-specific trait involved in extracellular matrix formation and cell-cell interaction.

Sun-screening bioactive compounds mycosporine-like amino acids in naturally occurring cyanobacterial biofilms: role in photoprotection.

AIMS: To investigate the occurrence of UV sunscreening biomolecules and their role in photoprotection in cyanobacterial biofilms growing in brightly lit habitats with high UV fluxes. METHODS AND RESULTS: High performance liquid chromatography with photodiode-array and mass spectrometry revealed the presence of mycosporine-like amino acids (MAAs) shinorine (λ(max) 334 nm, m/z 333), porphyra-334 (λ(max) 334 nm, m/z 347), mycosporine-glycine (λ(max) 310 nm, m/z 246) and palythinol (λ(max) 332 nm, m/z 303). Two unknown MAAs with λ(max) at 320 (m/z 289) and 329 nm (m/z 318) were also found. Biosynthesis of MAAs was found to increase with increase in exposure time under UV radiation. The MAAs from biofilms showed efficient radical scavenging activity as well as photoprotective potential on the survival of UV-treated Escherichia coli cells. CONCLUSIONS: Biosynthesis of photoprotectants is an important mechanism to prevent photodamage in Cyanobacteria. UV-induction and photoprotective function of MAAs may facilitate them to perform important ecological functions under harsh environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: There are very few reports on qualitative and quantitative characterization of different MAAs in cyanobacterial biofilms. Due to strong UV absorption and photoprotective function, MAAs may be used as an active ingredient in cosmetic and other pharmaceutical industries.

Seasonal Changes in Mycosporine-Like Amino Acid Production Rate with Respect to Natural Phytoplankton Species Composition.

After in situ incubation at the site for a year, phytoplanktons in surface water were exposed to natural light in temperate lakes (every month); thereafter, the net production rate of photoprotective compounds (mycosporine-like amino acids, MAAs) was calculated using (13)C labeled tracer. This is the first report describing seasonal variation in the net production rate of individual MAAs in temperate lakes using a compound-specific stable isotope method. In the mid-latitude region of the Korean Peninsula, UV radiation (UVR) usually peaks from July to August. In Lake Paldang and Lake Cheongpyeong, diatoms dominated among the phytoplankton throughout the year. The relative abundance of Cyanophyceae (Anabaena spiroides) reached over 80% during July in Lake Cheongpyeong. Changes in phytoplankton abundance indicate that the phytoplankton community structure is influenced by seasonal changes in the net production rate and concentration of MAAs. Notably, particulate organic matter (POM) showed a remarkable change based on the UV intensity occurring during that period; this was because of the fact that cyanobacteria that are highly sensitive to UV irradiance dominated the community. POM cultured in Lake Paldang had the greatest shinorine (SH) production rate during October, i.e., 83.83 ± 10.47 fgC·L(-1)·h(-1). The dominance of diatoms indicated that they had a long-term response to UVR. Evaluation of POM cultured in Lake Cheongpyeong revealed that there was an increase in the net MAA production in July (when UVR reached the maximum); a substantial amount of SH, i.e., 17.62 ± 18.34 fgC·L(-1)·h(-1), was recorded during this period. Our results demonstrate that both the net production rate as well as the concentration of MAAs related to photoinduction depended on the phytoplankton community structure. In addition, seasonal changes in UVR also influenced the quantity and production of MAAs in phytoplanktons (especially Cyanophyceae).

Contrasting patterns of MAAs accumulation in two populations of the copepod Boeckella gracilipes.

The bio-accumulation of mycosporine-like amino acids (MAAs) is common in planktonic copepods that inhabit environments exposed to high levels of solar radiation. MAAs accumulation in copepods can be affected both by extrinsic (environmental) and intrinsic factors (local adaptation, genotype, etc.). Laboratory experiments were performed to study the bio-accumulation of MAAs in two geographically-isolated populations of Boeckella gracilipes from a mountain and a piedmont lake of North Patagonia. We performed two series of 10-day incubations of B. gracilipes from the different lakes applying two radiation conditions (PAR + UVR and darkness), at five different temperatures (5 to 20 °C) and providing a MAA-free flagellate as food. We assumed that differences in final MAAs concentrations between copepod populations should be exclusively due to environmental factors, and that any difference in the patterns of MAAs accumulation should exclusively arise from differences in MAAs concentration at the time of collection. MAAs concentration was three fold higher in B. gracilipes from Lake Verde than in copepods from the Lake Morenito. The MAAs suite was dominated (∼90%) by a combination of porphyra-334 and mycosporine-glycine in copepods from Lake Verde, and porphyra-334 and MAA-332 in those from Lake Morenito. Two exclusive MAA compounds were identified, mycosporine-glycine in copepods from Lake Verde and shinorine in the copepod population from Lake Morenito. Laboratory experiments showed that: (i) exposure to PAR + UVR stimulated the accumulation of MAAs in both copepod populations; (ii) temperature affected the response of MAAs and, remarkably, low temperatures stimulated MAAs accumulation even in dark incubations, (iii) the response to radiation and temperature in MAAs accumulation was more pronounced in the population with low initial MAAs than in the population with high initial MAAs concentrations. The differences in intrinsic factors between B. gracilipes populations, such as local adaptation to contrasting UV and temperature scenarios, among others, appear to play an important role in determining levels and patterns of MAAs accumulation in B. gracilipes.

[Enzymatic production of 1,4-cyclohexanedimethylamine].

1,4-cyclohexanedimethylamine (1,4-BAC) is an important monomer for bio-based materials, it finds wide applications in various fields including organic synthesis, medicine, chemical industry, and materials. At present, its synthesis primarily relies on chemical method, which suffer from issues such as expensive metal catalyst, harsh reaction conditions, and safety risks. Therefore, it is necessary to explore greener alternatives for its synthesis. In this study, a two-bacterium three-enzyme cascade conversion pathway was successfully developed to convert 1,4-cyclohexanedicarboxaldehyde to 1,4-cyclohexanedimethylamine. This pathway used Escherichia coli derived aminotransferase (EcTA), Saccharomyces cerevisiae derived glutamate dehydrogenase (ScGlu-DH), and Candida boidinii derived formate dehydrogenase (CbFDH). Through structure-guided protein engineering, a beneficial mutant, EcTAF91Y, was obtained, exhibiting a 2.2-fold increase in specific activity and a 1.9-fold increase in kcat/Km compared to that of the wild type. By constructing recombinant strains and optimizing reaction conditions, it was found that under the optimal conditions, a substrate concentration of 40 g/L could produce (27.4±0.9) g/L of the product, corresponding to a molar conversion rate of 67.5%±2.1%.

Spermidine is a morphogenetic determinant for cell fate specification in the male gametophyte of the water fern Marsilea vestita.

Here, we show that the polyamine spermidine plays a key role as a morphogenetic determinant during spermatid development in the water fern Marsilea vestita. Spermidine levels rise first in sterile jacket cells and then increase dramatically in spermatogenous cells as the spermatids mature. RNA interference and drug treatments were employed to deplete spermidine in the gametophyte at different stages of gametogenesis. Development in spermidine-depleted gametophytes was arrested before the completion of the last round of cell divisions. In spermidine-depleted spermatogenous cells, chromatin failed to condense properly, basal body positioning was altered, and the microtubule ribbon was in disarray. When cyclohexylamine, a spermidine synthase (SPDS) inhibitor, was added at the start of spermatid differentiation, the spermatid nuclei remained round, centrin failed to localize into basal bodies, thus blocking basal body formation, and the microtubule ribbon was completely abolished. In untreated gametophytes, spermidine made in the jacket cells moves into the spermatids, where it is involved in the unmasking of stored SPDS mRNAs, leading to substantial spermidine synthesis in the spermatids. We found that treating spores directly with spermidine or other polyamines was sufficient to unmask a variety of stored mRNAs in gametophytes and arrest development. Differences in patterns of transcript distribution after these treatments suggest that specific transcripts reside in different locations in the dry spore; these differences may be linked to the timing of unmasking and translation for that mRNA during development.

Beyond ferrostatin-1: a comprehensive review of ferroptosis inhibitors.

Ferroptosis is an iron-catalysed form of regulated cell death, which is critically dependent on phospholipid peroxidation of cellular membranes. Ferrostatin 1 was one of the first synthetic radical-trapping antioxidants (RTAs) reported to block ferroptosis and it is widely used as reference compound. Ferroptosis has been linked to multiple diseases and the use of its inhibitors could have therapeutic potential. Although, novel biochemical pathways provide insights for different pharmacological targets, the use of lipophilic RTAs to block ferroptosis remains superior. In this Review, we provide a comprehensive overview of the different classes of ferroptosis inhibitors, focusing on endogenous and synthetic RTAs. A thorough analysis of their chemical, pharmacokinetic, and pharmacological properties and potential for in vivo use is provided.

Distribution of mycosporine-like amino acids along a surface water meridional transect of the Atlantic.

The composition and abundance of mycosporine-like amino acids (MAAs) were investigated in the surface waters along a 13,000-km meridional transect (52° N to 45° S) in the Atlantic Ocean (Atlantic Meridional Transect programme: Cruise AMT 18: 4/10/2008-10/11/2008). MAAs were ubiquitous along the transect, although the composition of the MAAs was variable. Highest concentrations were in the far south (below 40° S; MAA >1 µg L(-1)) and in north subtropical equatorial region (NER: 0-25° N; MAA up to 0.8 µg L(-1)). Highest MAA relative to chlorophyll-a occurred in the NER (MAA/chl-a ratio between 2 and 5). MAA/chl-a significantly correlated with the preceding month's mean daily UV dose and with UV-B/UV-A. In the far south, high MAA concentrations coincided with high phytoplankton biomass, high nutrients and a deep mixed layer associated with the austral spring. Here, the phytoplankton community was dominated by micro- and nano-eukaryotes. At the NER, the high MAA/chl-a coincided with low nutrient concentrations, a shallow mixed layer depth (20-70 m) and to a lesser extent to a shallow nitracline (40-90 m). Here, the phytoplankton consisted primarily of picophytoplankton (0-0.2 µm), dominated by the pico-cyanobacteria Synechococcus sp. and Prochlorococcus sp. and by the nitrogen fixing filamentous cyanobacterium Trichodesmium. The low nitrate concentrations (<0.1 µmol L(-1)) at the NER suggest that nitrogen fixation was required for MAA production. Specific MAAs could not easily be assigned to particular groups of phytoplankton and we could not rule out the possibility that MAAs were associated with symbiotic cyanobacteria contained within heterotrophic dinoflagellates or diatoms.