Ethical considerations for the age of non-governmental space exploration.

Mounting ambitions and capabilities for public and private, non-government sector crewed space exploration bring with them an increasingly diverse set of space travelers, raising new and nontrivial ethical, legal, and medical policy and practice concerns which are still relatively underexplored. In this piece, we lay out several pressing issues related to ethical considerations for selecting space travelers and conducting human subject research on them, especially in the context of non-governmental and commercial/private space operations.

A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation.

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.

Heterologous constitutive production of short-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2440: the involvement of IbpA inclusion body protein.

Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain à la carte materials with tailored physicochemical properties. To this end, we used the model medium-chain-length-PHA producing bacterium, P. putida KT2440 as a chassis, which is characterized by its metabolic versatility and stress tolerance. Different PHA biosynthetic modules were assembled in expression plasmids using the Golden gate/MoClo modular assembly technique to implement an orthogonal short-chain-lengh-PHA (scl-PHA) switch in a "deaf" PHA mutant. This was specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. We generated a panel of engineered approaches carrying the genes from Rhodospirillum rubrum, Cupriavidus necator and Pseudomonas pseudoalcaligenes, demonstrating that diverse scl-PHAs can be constitutively produced in the chassis strain to varying yields from 23% to 84% PHA/CDW. Co-feeding assays of the most promising engineered strain harboring the PHA machinery from C. necator resulted to a panel of PHBV from 0.6% to 19% C5 monomeric incorporation. Chromosomally integrated PHA machineries with high PhaCCn synthase dosage successfully resulted in 68% PHA/CDW production. Interestingly, an inverse relationship between PhaC synthase dosage and granule size distribution was demonstrated in the heterologous host. In this vein, it is proposed the key involvement of inclusion body protein IbpA to the heterologous production of tailored PHA in P. putida KT2440.

Synthetic Control of Metabolic States in Pseudomonas putida by Tuning Polyhydroxyalkanoate Cycle.

Polyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a dynamic cycle linked to the central carbon metabolism, which modulates numerous and diverse bacterial processes, such as stress endurance, pathogenesis, and persistence. Here, we analyze the role of the PHA cycle in conferring robustness to the model bacterium P. putida KT2440. To assess the effect of this cycle in the cell, we began by constructing a PHA depolymerase (PhaZ) mutant strain that had its PHA cycle blocked. We then restored the flux through the cycle in the context of an engineered library of P. putida strains harboring differential levels of PhaZ. High-throughput phenotyping analyses of this collection of strains revealed significant changes in response to PHA cycle performance impacting cell number and size, PHA accumulation, and production of extracellular (R)-hydroxyalkanoic acids. To understand the metabolic changes at the system level due to PHA turnover, we contextualized these physiological data using the genome-scale metabolic model iJN1411. Model-based predictions suggest successive metabolic steady states during the growth curve and an important carbon flux rerouting driven by the activity of the PHA cycle. Overall, we demonstrate that modulating the activity of the PHA cycle gives us control over the carbon metabolism of P. putida, which in turn will give us the ability to tailor cellular mechanisms driving stress tolerance, e.g., defenses against oxidative stress, and any potential biotechnological applications. IMPORTANCE Despite large research efforts devoted to understanding the flexible metabolism of Pseudomonas beyond the role of key regulatory players, the metabolic basis powering the dynamic control of its biological fitness under disturbance conditions remains largely unknown. Among other metabolic hubs, the so-called PHA cycle, involving simultaneous synthesis and degradation of PHAs, is emerging as a pivotal metabolic trait powering metabolic robustness and resilience in this bacterial group. Here, we provide evidence suggesting that metabolic states in Pseudomonas can be anticipated, controlled, and engineered by tailoring the flux through the PHA cycle. Overall, our study suggests that the PHA cycle is a promising metabolic target toward achieving control over bacterial metabolic robustness. This is likely to open up a broad range of applications in areas as diverse as pathogenesis and biotechnology.

Crystal Structure and Solid-State Packing of 4-Chloro-5H-1,2,3-dithiazol-5-one and 4-Chloro-5H-1,2,3-dithiazole-5-thione.

The crystal structure and solid-state packing of 4-chloro-5H-1,2,3-dithiazol-5-one and two polymorphs of 4-chloro-5H-1,2,3-dithiazole-5-thione were analyzed and compared to structural data of similar systems. These five-membered S,N-rich heterocycles are planar with considerable bond localization. All three structures demonstrate tight solid-state packing without voids which is attributed to a rich network of short intermolecular electrostatic contacts. These include Sδ+…Nδ-, Sδ+…Oδ-, Sδ+…Clδ- and Sδ+…Sδ- interactions that are well within the sum of their van der Waals radii (∑VDW). B3LYP, BLYP, M06, mPW1PW, PBE and MP2 were employed to calculate their intramolecular geometrical parameters, the Fukui condensed functions to probe their reactivity, the bond order, Bird Index and NICS(1) to establish their aromaticity.

Engineering Native and Synthetic Pathways in Pseudomonas putida for the Production of Tailored Polyhydroxyalkanoates.

Growing environmental concern sparks renewed interest in the sustainable production of (bio)materials that can replace oil-derived goods. Polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in the central metabolism of producer bacteria, as they act as dynamic reservoirs of carbon and reducing equivalents. PHAs continue to attract industrial attention as a starting point toward renewable, biodegradable, biocompatible, and versatile thermoplastic and elastomeric materials. Pseudomonas species have been known for long as efficient biopolymer producers, especially for medium-chain-length PHAs. The surge of synthetic biology and metabolic engineering approaches in recent years offers the possibility of exploiting the untapped potential of Pseudomonas cell factories for the production of tailored PHAs. In this article, an overview of the metabolic and regulatory circuits that rule PHA accumulation in Pseudomonas putida is provided, and approaches leading to the biosynthesis of novel polymers (e.g., PHAs including nonbiological chemical elements in their structures) are discussed. The potential of novel PHAs to disrupt existing and future market segments is closer to realization than ever before. The review is concluded by pinpointing challenges that currently hinder the wide adoption of bio-based PHAs, and strategies toward programmable polymer biosynthesis from alternative substrates in engineered P. putida strains are proposed.

Modulation of calcineurin activity in Aspergillus nidulans: the roles of high magnesium concentrations and of transcriptional factor CrzA.

A proper response to elevated extracellular calcium levels helps to most organisms to keep this secondary messenger under strict control, thereby preventing inadequate activation or inhibition of many regulatory activities into cells. In fungi, the calcineurin responsive zinc-finger Crz1/CrzA transcription factor transduces calcium signaling to gene expression. In Aspergillus nidulans, absence of CrzA activity leads to alkaline pH sensitivity and loss of tolerance to high levels of extracellular calcium. Disruption of calcium uptake mechanisms or the presence of high levels of Mg2+ partially suppresses this calcium-sensitive phenotype of null crzA strain. The effects of Mg2+ on CrzA phosphorylation and perturbations that reduce calcineurin phosphatase activity on CrzA demonstrate that the calcium sensitive phenotype of null crzA strain is a consequence of up-regulated calcineurin activity under calcium-induced conditions.

Redox Active Quinoidal 1,2,4-Benzotriazines.

Modifying the para-quinonimine 1,3-diphenyl-1,2,4-benzotriazin-7(1 H)-one (2a) ( E1/2-1/0 -1.20 V), by replacing the N1-phenyl by pentafluorophenyl, the C3-phenyl by trifluoromethyl, or the C7 carbonyl by ylidenemalononitrile, led to improved electron affinities as determined by cyclic voltammetry and computational studies. Combining structural changes further improved electron accepting abilities: the most electron deficient analogues ( E1/2-1/0 ∼ -0.65 V) involved combining the ylidenemalononitrile groups at C7 with the trifluoromethyl groups at C3. 1,2,5-Thiadiazolo fusion at C5-C6 did not affect the redox behavior but did enhance the UV-vis absorption profile. During the synthesis of the thiadiazolo analogues, 1,4-thiazino-fused analogues 6 were obtained in low yield, which thermally ring contract to the triazafluoranthenones 7. Compounds are fully characterized, and X-ray data are provided for selected analogues.

A hexameric [MnNa6] wheel based on [MnO]7+ sub-units.

A novel hexameric [MnNa6] wheel-like aggregate consisting of [MnO] triangles is reported. It is the second highest nuclearity oxime-based Mn cluster, the largest member of the recently-developed family of molecular oligomers based on [MnO] triangles, and the only one with a wheel-like metal topology.

Nimesulide Silver Metallodrugs, Containing the Mitochondriotropic, Triaryl Derivatives of Pnictogen; Anticancer Activity against Human Breast Cancer Cells.

Novel silver(I) metallo-drugs of the nonsteroidal anti-inflammatory drug nimesulide (nim) and the mitochondriotropic triaryl derivatives of pnictogen ligands (tpE, E = P (tpp, tptp, or totp), As (tpAs), Sb (tpSb)) with the formulas {[Ag(nim) (tpp)2]DMF} (1), [Ag(nim) (tptp)2] (2), [Ag(nim) (totp)] (3), [Ag(nim) (tpAs)2] (4), and [Ag(nim) (tpSb)3] (5) ((tpp = triphenyphosphine, tptp = tri(p-tolyl)phosphine, totp = tri(o-tolyl)phosphine, tpAs = triphenylarsine, tpSb = triphenylantimony, and DMF = dimethylformamide) were synthesized and characterized by melting point, vibrational spectroscopy (mid-Fourier transform IR), (1)H NMR, UV-visible spectroscopic techniques, and X-ray crystallography. The in vitro cytotoxic activity of 1-5 against human breast adenocarcinoma cancer cell lines: MCF-7 (estrogen receptor (ER) positive) and MDA-MB-231 (ER negative) was determined. The genotoxicity on normal human fetal lung fibroblast cells (MRC-5) caused by 1-5 was evaluated by fluorescence microscopy. The absence of micronucleus in MRC-5 cells confirms the in vitro non toxicity behavior of the compounds. Because of the morphology of the cells, an apoptotic pathway was concluded for the cell death. The apoptotic pathway, especially though the mitochondrion damage, was confirmed by DNA fragmentation, cell cycle arrest, and permeabilization of the mitochondrial membrane tests. The molecular mechanism of action of 1-5 was further studied by (i) the binding affinity of 1-5 toward the calf thymus (CT) DNA, (ii) the inhibitory activity of 1-5 against lipoxygenase (an enzyme that oxidizes polyunsaturated fatty acids to leukotrienes or prostaglandins), and (iii) the catalytic activity of 1-5 on the oxidation of linoleic acid (an acid that partakes in membrane fluidity, membrane enzyme activities, etc.) to hyperoxolinoleic acid by oxygen.

New applications for known drugs: Human glycogen synthase kinase 3 inhibitors as modulators of Aspergillus fumigatus growth.

Invasive aspergillosis (IA) is one of the most severe forms of fungi infection. IA disease is mainly due to Aspergillus fumigatus, an air-borne opportunistic pathogen. Mortality rate caused by IA is still very high (50-95%), because of difficulty in early diagnostics and reduced antifungal treatment options, thus new and efficient drugs are necessary. The aim of this work is, using Aspergillus nidulans as non-pathogen model, to develop efficient drugs to treat IA. The recent discovered role of glycogen synthase kinase-3 homologue, GskA, in A. fumigatus human infection and our previous experience on human GSK-3 inhibitors focus our attention on this kinase as a target for the development of antifungal drugs. With the aim to identify effective inhibitors of colonial growth of A. fumigatus we use A. nidulans as an accurate model for in vivo and in silico studies. Several well-known human GSK-3ß inhibitors were tested for inhibition of A. nidulans colony growth. Computational tools as docking studies and binding site prediction was used to explain the different biological profile of the tested inhibitors. Three of the five tested hGSK3ß inhibitors are able to reduce completely the colonial growth by covalent bind to the enzyme. Therefore these compounds may be useful in different applications to eradicate IA.

The Suppression of Columnar π-Stacking in 3-Adamantyl-1-phenyl-1,4-dihydrobenzo[e][1,2,4]triazin-4-yl.

3-Adamantyl-1-phenyl-1,4-dihydrobenzo[e][1,2,4]triazin-4-yl (4) crystallizes as chains of radicals where the spin bearing benzotriazinyl moieties are isolated from each other. Magnetic susceptibility studies in the 5-300 K temperature region indicate that radical 4 demonstrates typical paramagnetic behavior stemming from non-interacting S = ½ spins.

Oxidation of Tetraphenylhexaazaanthracene: Accessing a Scissor Dimer of a 16π Biscyanine.

Tetraphenylhexaazaanthracene (TPHA), a fluorescent zwitterionic biscyanine with a closed-shell singlet ground state, on treatment with manganese dioxide or phenyliodine bis(trifluoroacetate) (PIFA), undergoes oxidative dimerization to give a near-zero dipole scissor 5,5'-dimer DI-TPHA. Both acene components of the new dimer DI-TPHA maintain their biscyanine closed-shell singlet ground state motifs, as judged by analysis of both single crystal X-ray crystallographic and density functional theory computational studies; however, unlike TPHA, DI-TPHA is only very weakly fluorescent.

Magnetic "Molecular Oligomers" Based on Decametallic Supertetrahedra: A Giant Mn49 Cuboctahedron and its Mn25Na4 Fragment.

Two nanosized Mn49 and Mn25Na4 clusters based on analogues of the high-spin (S=22) [Mn(III)6Mn(II)4(µ4-O)4](18+) supertetrahedral core are reported. Mn49 and Mn25Na4 complexes consist of eight and four decametallic supertetrahedral subunits, respectively, display high virtual symmetry (O(h)), and are unique examples of clusters based on a large number of tightly linked high nuclearity magnetic units. The complexes also have large spin ground-state values (Mn49: S=61/2; Mn25Na4: S=51/2) with the Mn49  cluster displaying single-molecule magnet (SMM) behavior and being the second largest reported homometallic SMM.

The Reaction of DABCO with 4-Chloro-5H-1,2,3-dithiazoles: Synthesis and Chemistry of 4-[N-(2-Chloroethyl)piperazin-1-yl]-5H-1,2,3-dithiazoles.

N-(4-Chloro-5H-1,2,3-dithiazol-5-ylidene)anilines react with DABCO in hot PhCl to give N-{4-[N-(2-chloroethyl)piperazin-1-yl]-5H-1,2,3-dithiazol-5-ylidene}anilines in high yields (70-92%). The reaction also works with 4-chloro-5H-1,2,3-dithiazol-5-one and -thione, giving the corresponding products in 85% and 76% yields, respectively. While the reaction of several (4-chloro-5H-1,2,3-dithiazol-5-ylidene)methanes with DABCO failed to give {4-[N-(2-chloroethyl)piperazin-1-yl]-5H-1,2,3-dithiazol-5-ylidene}methanes, these can be prepared in moderate yields via classical cycloaddition-retrocycloaddition strategies from 4-[N-(2-chloroethyl)piperazin-1-yl]-5H-1,2,3-dithiazole-5-thione. The 2-chloroethyl moiety on selected dithiazoles was also modified without cleavage of the 1,2,3-dithiazole by reaction with various nucleophiles, giving access to 4-[N-(2-substituted)piperazin-1-yl]-5H-1,2,3-dithiazoles in moderate to high yields.

Tetraphenylhexaazaanthracenes: 16π Weakly Antiaromatic Species with Singlet Ground States.

Tetraphenylhexaazaanthracene, TPHA-1, is a fluorescent zwitterionic biscyanine with a closed-shell singlet ground state. TPHA-1 overcomes its weak 16π antiaromaticity by partitioning its π system into 6π positive and 10π negative cyanines. The synthesis of TPHA-1 is low yielding and accompanied by two analogous TPHA isomers: the deep red, non-charge-separated, quinoidal TPHA-2, and the deep green TPHA-3 that partitions into two equal but oppositely charged 8π cyanines. The three TPHA isomers are compared.

Synthesis of N-Aryl-3,5-dichloro-4H-1,2,6-thiadiazin-4-imines from 3,4,4,5-Tetrachloro-4H-1,2,6-thiadiazine.

Condensation of 3,4,4,5-tetrachloro-4H-1,2,6-thiadiazine with a range of anilines gave 22 N-aryl-3,5-dichloro-4H-1,2,6-thiadiazin-4-imines in 43-96% yields. The scope and limitations of this condensation are briefly investigated. Furthermore, mono- and bis-substitution of the C-3 and C-5 chlorines of 3,5-dichloro-N-phenyl-4H-1,2,6-thiadiazin-4-imine by amine and alkoxide nucleophiles is explored. Finally, Stille coupling chemistry is used to prepare several N-phenyl-3,5-diaryl-4H-1,2,6-thiadiazin-4-imines.

Synthesis of fused 1,2,4-dithiazines and 1,2,3,5-trithiazepines.

Reacting (Z)-N-(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-1H-pyrazol-5-amines 5 with Et2NH and then with concd H2SO4 gives 5H-pyrazolo[3,4-e][1,2,4]dithiazine-3-carbonitriles 7 in good yields (74-85%) and 6H-pyrazolo[3,4-f][1,2,3,5]trithiazepine-4-carbonitriles 9 as minor products (0-6%). Furthermore, the 1,3-dimethylpyrazole analogue 5a was transformed into the dithiazine 7a in two discrete steps, allowing the isolation of a disulfide intermediate (Z)-2-[(diethylamino)disulfan-yl]-2-[(1H-pyrazol-5-yl)imino]acetonitrile (8a). The one-pot, two-step reaction also worked with electron-rich hydroxy- and methoxy-substituted anilines. Thermolysis of the pyrazolo[3,4-e][1,2,4]dithiazines 7 gave the ring-contracted 1H-pyrazolo[3,4-d]thiazole-5-carbonitriles 6 (94-100%). With active sulfur, 1,3-dimethyl-5H-pyrazolo[3,4-e][1,2,4]dithiazine-3-carbonitrile (7a) gave 1,3-dimethyl-6H-pyrazolo[3,4-f][1,2,3,5]trithiazepine-4-carbonitrile (9a), but on prolonged reaction times, it gave 5,7-dimethyl-5H-[1,2,3]dithiazolo[4,5-b]pyrazolo[3,4-e][1,4]thiazine (13). Finally, in the absence of acid, heating a solution of (Z)-2-[(diethylamino)disulfanyl]-2-[(1,3-dimethyl-1H-pyrazol-5-yl)imino]acetonitrile (8a) gave 4,6,10,12-tetramethyl-6H-pyrazolo[3,4-f]pyrazolo[3',4':4,5]pyrimido[6,1-d][1,2,3,5]trithiazepine-8,12b(10H)-dicarbonitrile (19) (67%).