[Developmental toxicity of Cry1Ab protein in the embryonic stem-cell model].

OBJECTIVE: To evaluate the developmental toxicity of Cry1Ab protein by studying its effects on cell proliferation and differentiation ability using a developmental toxicity assessment model based on embryonic stem-cell. METHODS: Cry1Ab protein was tested in seven dose groups (31.25, 62.50, 125.00, 250.00, 320.00, 1 000.00, and 2 000.00 µg/L) on mouse embryonic stem cells D3 (ES-D3) and 3T3 mouse fibroblast cells, with 5-fluorouracil (5-FU) used as the positive control and phosphate buffer saline (PBS) as the solvent control. Cell viability was detected by CCK-8 assay to calculate the 50% inhibitory concentration (IC50) of the test substance for different cells. Additionally, Cry1Ab protein was tested in five dose groups (125.00, 250.00, 320.00, 1 000.00, and 2 000.00 µg/L) on ES-D3 cells, with PBS as the solvent control and 5-FU used for model validation. After cell treatment, cardiac differentiation was induced using the embryonic bodies (EBs) culture method. The growth of EBs was observed under a microscope, and their diameters on the third and fifth days were measured. The proportion of EBs differentiating into beating cardiomyocytes was recorded, and the 50% inhibition concentration of differentiation (ID50) was calculated. Based on a developmental toxicity discrimination function, the developmental toxicity of the test substances was classified. Furthermore, at the end of the culture period, mRNA expression levels of cardiac differentiation-related markers (Oct3/4, GATA-4, Nkx2.5, and ß-MHC) were quantitatively detected using real-time quantitative polymerase chain reaction (qPCR) in the collected EBs samples. RESULTS: The IC50 of 5-FU was determined as 46.37 µg/L in 3T3 cells and 32.67 µg/L in ES-D3 cells, while the ID50 in ES-D3 cells was 21.28 µg/L. According to the discrimination function results, 5-FU was classified as a strong embryotoxic substance. There were no statistically significant differences in cell viability between different concentrations of Cry1Ab protein treatment groups and the control group in both 3T3 cells and ES-D3 cells (P>0.05). Moreover, there were no statistically significant differences in the diameter of EBs on the third and fifth days, as well as their morphology, between the Cry1Ab protein treatment groups and the control group (P>0.05). The cardiac differentiation rate showed no statistically significant differences between different concentrations of Cry1Ab protein treatment groups and the control group (P>0.05). 5-FU significantly reduced the mRNA expression levels of ß-MHC, Nkx2.5, and GATA-4 (P < 0.05), showing a dose-dependent trend (P < 0.05), while the mRNA expression levels of the pluripotency-associated marker Oct3/4 exhibited an increasing trend (P < 0.05). However, there were no statistically significant differences in the mRNA expression levels of mature cardiac marker ß-MHC, early cardiac differentiation marker Nkx2.5 and GATA-4, and pluripotency-associated marker Oct3/4 between the Cry1Ab protein treatment groups and the control group (P>0.05). CONCLUSION: No developmental toxicity of Cry1Ab protein at concentrations ranging from 31.25 to 2 000.00 µg/L was observed in this experimental model.

Continuous H-B-E fermentation by Clostridium carboxidivorans: CO vs syngas.

Leveraging renewable carbon-based resources for energy and chemical production is a promising approach to decrease reliance on fossil fuels. This entails a thermo/biotechnological procedure wherein bacteria, notably Clostridia, ferment syngas, converting CO or CO2 + H2 into Hexanol, Butanol and Ethanol (H-B-E fermentation). This work reports of Clostridium carboxidivorans performance in a stirred tank reactor continuously operated with respect to the gas and the cell/liquid phases. The primary objective was to assess acid and solvent production at pH 5.6 by feeding pure CO or synthetic syngas under gas flow differential conditions. Fermentation tests were conducted at four different dilution rates (DL) of the fresh medium in the range 0.034-0.25 h-1. The fermentation pathways of C. carboxidivorans were found to be nearly identical for both CO and syngas, with consistent growth and metabolite production at pH 5.6 within a range of dilution rates. Wash-out conditions were observed at a DL of 0.25 h-1 regardless of the carbon source. Ethanol was the predominant solvent produced, but a shift towards butanol production was observed with CO as the substrate and towards hexanol production with synthetic syngas. In particular, the maximum cell concentration (0.5 gDM/L) was obtained with pure CO at DL 0.05 h-1; the highest solvent productivity (60 mg/L*h of total solvent) was obtained at DL 0.17 h-1 by using synthetic syngas as C-source. The findings highlight the importance of substrate composition and operating conditions in syngas fermentation processes. These insights contribute to the optimization of syngas fermentation processes for biofuel and chemical production.

Genome-scale metabolic network model and phenome of solvent-tolerant Pseudomonas putida S12.

BACKGROUND: Pseudomonas putida S12 is a gram-negative bacterium renowned for its high tolerance to organic solvents and metabolic versatility, making it attractive for various applications, including bioremediation and the production of aromatic compounds, bioplastics, biofuels, and value-added compounds. However, a metabolic model of S12 has yet to be developed. RESULTS: In this study, we present a comprehensive and highly curated genome-scale metabolic network model of S12 (iSH1474), containing 1,474 genes, 1,436 unique metabolites, and 2,938 metabolic reactions. The model was constructed by leveraging existing metabolic models and conducting comparative analyses of genomes and phenomes. Approximately 2,000 different phenotypes were measured for S12 and its closely related KT2440 strain under various nutritional and environmental conditions. These phenotypic data, combined with the reported experimental data, were used to refine and validate the reconstruction. Model predictions quantitatively agreed well with in vivo flux measurements and the batch cultivation of S12, which demonstrated that iSH1474 accurately represents the metabolic capabilities of S12. Furthermore, the model was simulated to investigate the maximum theoretical metabolic capacity of S12 growing on toxic organic solvents. CONCLUSIONS: iSH1474 represents a significant advancement in our understanding of the cellular metabolism of P. putida S12. The combined results of metabolic simulation and comparative genome and phenome analyses identified the genetic and metabolic determinants of the characteristic phenotypes of S12. This study could accelerate the development of this versatile organism as an efficient cell factory for various biotechnological applications.

Development of a tadalafil transdermal formulation and evaluation of its ability to in vitro transdermal permeate using Strat-M® membrane.

Tadalafil (TDF) has low water solubility, high intestinal permeability and belongs to the Biopharmaceutics Classification System (BCS) Class II. Due to high intestinal permeability, only oral administration (tablets) and oral thin film formulations have been developed. Therefore, it is necessary to develop various formulations, such as external formulations and transdermal absorption formulations requested by patients. The purpose of this study is to improve the solubility and skin permeability of TDF, and to develop a novel transdermal formulation with secured stability over time. The research strategy is to determine solvents that will improve TDF solubility and to screen substances that will enhance TDF permeability. Skin penetration tests were simulated by using a Strat-M® membrane in Franz diffusion cell systems. The optimal formulation (F1, consisting of TDF/HDTMA-Br at a ratio of 1:10 [weight/weight] in DPG) observed the highest permeability compared to all formulations in PBS (pH 7.4). Changes in thermal property of F1 formulation was observed and maintained its stability over 12 months including drug content (µg/mL), appearance, pH, and permeation (µg/cm2). In conclusion, DPG played a supported role in improving both TDF solubilization and permeability, whereas HDTMA-Br played a key role in enhancing permeability. It is thought that these results will be supplemented in the future to conduct research and experiments on humans.

[Inhibition of GMA-induced ubiquitination process in 16HBE malignantly transformed cells on protein CD44 and MMP14].

OBJECTIVE: To observe the effect of the ubiquitination process on the expression of CD44 antigen(CD44) and matrix metalloproteinase-14(MMP14) in human bronchial epithelial(16HBE) malignantly transformed cells induced by glycidyl methacrylate(GMA). METHODS: Successfully resuscitated 16HBE cells were cultured using a final concentration of 8 µg/mL GMA as the treatment group and 1 µg/mL dimethyl sulfoxide as the solvent control group, each time stained for 72 h, and then stained again after an interval of 24 h. After repeating the staining three times, the cells were cultured in passages respectively. The 40th generation(P40) GMA-treated group and the same-generation solvent control group were subjected to soft agar colony formation assay and concanavalin A(ConA) agglutination test to confirm that the 40th generation of GMA-induced malignant transformed 16HBE cells possessed malignant transformed cell characteristics.5, 10, 20, 40, 60 µmol/L anacardic acid were used to inhibit the ubiquitination process of GMA-induced malignant transformed 16HBE cells. The protein expression of CD44 and MMP14 were detected by western blotting, while the transcript levels of CD44, MMP14, and TFAP2A were assessed by real-time fluorescence quantitative PCR(qPCR). RESULTS: (1) In the soft agar colony formation assay, the number of clones formed by the cells in the solvent control group was 22, and the number of clones created by the malignantly transformed cells in the GMA-treated group was 208. In the ConA agglutination test, the cells in the solvent control group were uniformly dispersed in ConA solution, and no obvious agglutination occurred for 30 min, whereas the cells in the GMA-treated group were agglutinated in the 5th min, and the agglutinated cells were larger and more rapidly agglutinated. The agglomerates were more significant and faster, and the sensitivity of agglutination was increased. (2) After differential inhibition of GMA-induced ubiquitination in malignantly transformed 16HBE cells, the expression levels of CD44 and MMP14 were reduced in GMA-induced malignantly transformed 16HBE cells compared with the control group(P<0.05). The transcript levels of MMP14 and CD44 decreased with increasing inhibitor concentration(P<0.05), and the transcript levels of the upstream transcription factor TFAP2A were also simultaneously reduced(P<0.05). CONCLUSION: Inhibition of the cellular ubiquitination process mediates the down-regulation of protein expression and transcriptional expression of CD44 and MMP14 in GMA-induced malignantly transformed 16HBE cells.

Fabrication of dissolving microneedles for transdermal delivery of protein and peptide drugs: polymer materials and solvent casting micromoulding method.

Proteins and peptides are rapidly developing pharmaceutical products and are expected to continue growing in the future. However, due to their nature, their delivery is often limited to injection, with drawbacks such as pain and needle waste. To overcome these limitations, microneedles technology is developed to deliver protein and peptide drugs through the skin. One type of microneedles, known as dissolving microneedles, has been extensively studied for delivering various proteins and peptides, including ovalbumin, insulin, bovine serum albumin, polymyxin B, vancomycin, and bevacizumab. This article discusses polymer materials used for fabricating dissolving microneedles, which are poly(vinylpyrrolidone), hyaluronic acid, poly(vinyl alcohol), carboxymethylcellulose, GantrezTM, as well as other biopolymers like pullulan and ulvan. The paper is focused solely on solvent casting micromoulding method for fabricating dissolving microneedles containing proteins and peptides, which will be divided into one-step and two-step casting micromoulding. Additionally, future considerations in the market plan for dissolving microneedles are discussed in this article.

Resolving binding pathways and solvation thermodynamics of plant hormone receptors.

Plant hormones are small molecules that regulate plant growth, development, and responses to biotic and abiotic stresses. They are specifically recognized by the binding site of their receptors. In this work, we resolved the binding pathways for eight classes of phytohormones (auxin, jasmonate, gibberellin, strigolactone, brassinosteroid, cytokinin, salicylic acid, and abscisic acid) to their canonical receptors using extensive molecular dynamics simulations. Furthermore, we investigated the role of water displacement and reorganization at the binding site of the plant receptors through inhomogeneous solvation theory. Our findings predict that displacement of water molecules by phytohormones contributes to free energy of binding via entropy gain and is associated with significant free energy barriers for most systems analyzed. Also, our results indicate that displacement of unfavorable water molecules in the binding site can be exploited in rational agrochemical design. Overall, this study uncovers the mechanism of ligand binding and the role of water molecules in plant hormone perception, which creates new avenues for agrochemical design to target plant growth and development.

Macromolecular condensation buffers intracellular water potential.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

The cryo-EM 3D image reconstruction of isolated Lethocerus indicus Z-discs.

The Z-disk of striated muscle defines the ends of the sarcomere, which repeats many times within the muscle fiber. Here we report application of cryoelectron tomography and subtomogram averaging to Z-disks isolated from the flight muscles of the large waterbug Lethocerus indicus. We use high salt solutions to remove the myosin containing filaments and use gelsolin to remove the actin filaments of the A- and I-bands leaving only the thin filaments within the Z-disk which were then frozen for cryoelectron microscopy. The Lethocerus Z-disk structure is similar in many ways to the previously studied Z-disk of the honeybee Apis mellifera. At the corners of the unit cell are positioned trimers of paired antiparallel F-actins defining a large solvent channel, whereas at the trigonal positions are positioned F-actin trimers converging slowly towards their (+) ends defining a small solvent channel through the Z-disk. These near parallel F-actins terminate at different Z-heights within the Z-disk. The two types of solvent channel in Lethocerus are similar in size compared to those of Apis which are very different in size. Two types of α-actinin crosslinks were observed between oppositely oriented actin filaments. In one of these, the α-actinin long axis is almost parallel to the F-actins it crosslinks. In the other, the α-actinins are at a small but distinctive angle with respect to the crosslinked actin filaments. The utility of isolated Z-disks for structure determination is discussed.

Degradative solvent extraction of cyanobacteria: From reaction kinetics to potential organic matter evolution mechanism.

This study proposed a new continuous lumped reaction kinetics model to accurately reveal the control mechanism of cyanobacteria at each stage of degradative solvent extraction and discussed the potential evolution mechanism of organic matter. Results showed that degradation solvent extraction successfully separated nitrogen and phosphorus from cyanobacteria. The solute has high carbon and volatile contents, is almost ash-free, and forms a phosphorus-rich residue. The lowest fitting degree of the continuous lumped reaction model kinetics was 94.5%, suggesting that this model worked well. The depolymerization of the residue dominated between 200 and 350 °C, whereas solute decomposition dominated at 400 °C. Nitrogen-containing compounds, which originate from protein decarboxylation or deamination to generate amides, are the main components of the solute, and amino acids react with reducing sugars to generate nitrogen heterocyclic compounds, which are useful for preparing nitrogen-containing chemicals.

Improved biotransformation of lignin-valorized vanillin into vanillylamine in a sustainable bioreaction medium.

Lignin is a critical biopolymer for creating a large number of highly valuable biobased compounds. Vanillin, one of lignin-derived aromatics, can be used to synthesize vanillylamine that is a key fine chemical and pharmaceutical intermediate. To produce vanillylamine, a productive whole-cell-catalyzed biotransformation of vanillin was developed in deep eutectic solvent - surfactant - H2O media. One newly created recombinant E. coli 30CA cells expressing ω-transaminase and L-alanine dehydrogenase was employed to transform 50 mM and 60 mM vanillin into vanillylamine in the yield of 82.2% and 8.5% under 40 °C, respectively. The biotransamination efficiency was enhanced by introducing surfactant PEG-2000 (40 mM) and deep eutectic solvent ChCl:LA (5.0 wt%, pH 8.0), and the highest vanillylamine yield reached 90.0% from 60 mM vanillin. Building an effective bioprocess was utilized for transamination of lignin-derived vanillin to vanillylamine with newly created bacteria in an eco-friendly medium, which had potential application for valorization of lignin to value-added compounds.

Extraction of Nannochloropsis Fatty Acids Using Different Green Technologies: The Current Path.

Nannochloropsis is a genus of microalgae widely recognized as potential sources of distinct lipids, particularly polyunsaturated fatty acids (PUFA). These may be obtained through extraction, which has conventionally been performed using hazardous organic solvents. To substitute such solvents with "greener" alternatives, several technologies have been studied to increase their extraction potential. Distinct technologies utilize different principles to achieve such objective; while some aim at disrupting the cell walls of the microalgae, others target the extraction per se. While some methods have been utilized independently, several technologies have also been combined, which has proven to be an effective strategy. The current review focuses on the technologies explored in the last five years to extract or increase extraction yields of fatty acids from Nannochloropsis microalgae. Depending on the extraction efficacy of the different technologies, distinct types of lipids and/or fatty acids are obtained accordingly. Moreover, the extraction efficiency may vary depending on the Nannochloropsis species. Hence, a case-by-case assessment must be conducted in order to ascertain the most suited technology, or tailor a specific one, to be applied to recover a particular fatty acid (or fatty acid class), namely PUFA, including eicosapentaenoic acid.

Rewiring metabolism of Clostridium thermocellum for consolidated bioprocessing of lignocellulosic biomass poplar to produce short-chain esters.

Consolidated bioprocessing (CBP) of lignocellulosic biomass uses cellulolytic microorganisms to enable enzyme production, saccharification, and fermentation to produce biofuels, biochemicals, and biomaterials in a single step. However, understanding and redirecting metabolisms of these microorganisms compatible with CBP are limited. Here, a cellulolytic thermophile Clostridium thermocellum was engineered and demonstrated to be compatible with CBP integrated with a Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment for conversion of hardwood poplar into short-chain esters with industrial use as solvents, flavors, fragrances, and biofuels. The recombinant C. thermocellum engineered with deletion of carbohydrate esterases and stable overexpression of alcohol acetyltransferases improved ester production without compromised deacetylation activities. These esterases were discovered to exhibit promiscuous thioesterase activities and their deletion enhanced ester production by rerouting the electron and carbon metabolism. Ester production was further improved up to 80-fold and ester composition could be modulated by deleting lactate biosynthesis and using poplar with different pretreatment severity.

Biodegradation of 2-methylpentane in fluid fine tailings amended with a mixture of iso-alkanes under sulfate-reducing conditions.

Anaerobic microorganisms in Canada Natural Upgrading Limited (CNUL) fluid fine tailings (FFT) are sustained by residual solvent hydrocarbons. Although FFT are methanogenic in nature, sulfate-reducing microorganisms represent a significant portion of FFT bacterial community. In this study, we examined biodegradation of three iso-alkanes (2-methylbutane, 2-methylpentane, and 3-methylpentane), representing major iso-alkanes in paraffinic solvent, in CNUL FFT under sulfate-reducing conditions. During ∼1100 days of incubation, only 2-methylpentane was degraded partially, whereas 2-methylbutane and 3-methylpentane were not degraded. During active degradation of 2-methylpentane, the bacterial community was dominated by Anaerolineaceae followed by Syntrophaceae, Peptococcaceae, Desulfobacteraceae, and Desulfobulbaceae. The archaeal community was co-dominated by acetoclastic (Methanosaetaceae) and hydrogenotrophic (Methanobacteriaceae) methanogens. This study underlines the limited capability of the microbial community indigenous to CNUL FFT in degrading recalcitrant iso-alkanes under sulfate-reducing conditions.

ZIF-8 as support for enhanced stability of immobilized lipase used with a thermoresponsive switchable solvent to simplify the microalgae-to-biodiesel process.

The zeolitic imidazole framework (ZIF)- 8 was tested as a support to enhance the stability of immobilized lipase. The lipase immobilized on ZIF-8, through surface attachment and encapsulation, was used for the simultaneous cell disruption and oil extraction from untreated, wet microalgal paste. The successful attachment of the enzyme to ZIF-8 was confirmed via Fourier-transform infrared spectroscopy. The attachment of the enzyme did not significantly affect the crystallinity or morphology of ZIF-8 crystals. The encapsulated lipase@ZIF-8 system showed higher stability than the adsorbed system, due to its reduced vulnerability to leaching. After five cycles, the encapsulated lipase@ZIF-8 retained 32% of its initial activity, whereas, for the adsorbed lipase@ZIF-8, it reduced to 21%. An increase in methanol amount greater than 0.2 mL was shown to have a negative effect on enzyme activity. The fatty acid methyl ester yield increased significantly with an increase in the extraction- duration (up to 3 h), after which the effect faded until 5 h, after which the equilibrium yield was reached. Changing the composition of the thermoresponsive switchable solvent (TSS) showed that a higher FAME yield could be achieved by increasing the percentages of Ionic Liquid (IL) and polypropylene glycol and reducing the water percentage. Further studies are needed to optimize the TSS composition and its effects on the process.

Characterization and Application of a Novel Glucose Dehydrogenase with Excellent Organic Solvent Tolerance for Cofactor Regeneration in Carbonyl Reduction.

An efficient cofactor regeneration system has been developed to provide a hydride source for the preparation of optically pure alcohols by carbonyl reductase-catalyzed asymmetric reduction. This system employed a novel glucose dehydrogenase (BcGDH90) from Bacillus cereus HBL-AI. The gene encoding BcGDH90 was found through the genome-wide functional annotation. Homology-built model study revealed that BcGDH90 was a homo-tetramer, and each subunit was composed of ßD-αE-αF-αG-ßG motif, which was responsible for substrate binding and tetramer formation. The gene of BcGDH90 was cloned and expressed in Escherichia coli. The recombinant BcGDH90 exhibited maximum activity of 45.3 U/mg at pH 9.0 and 40 °C. BcGDH90 showed high stability in a wide pH range of 4.0-10.0 and was stable after the incubation at 55 °C for 5 h. BcGDH90 was not a metal ion-dependent enzyme, but Zn2+ could seriously inhibit its activity. BcGDH90 displayed excellent tolerance to 90% of acetone, methanol, ethanol, n-propanol, and isopropanol. Furthermore, BcGDH90 was applied to regenerate NADPH for the asymmetric biosynthesis of (S)-(+)-1-phenyl-1,2-ethanediol ((S)-PED) from hydroxyacetophenone (2-HAP) with high concentration, which increased the final efficiency by 59.4%. These results suggest that BcGDH90 is potentially useful for coenzyme regeneration in the biological reduction.

Insights into the susceptibility of Pseudomonas putida to industrially relevant aromatic hydrocarbons that it can synthesize from sugars.

Pseudomonas putida DOT-T1E is a highly solvent tolerant strain for which many genetic tools have been developed. The strain represents a promising candidate host for the synthesis of aromatic compounds-opening a path towards a green alternative to petrol-derived chemicals. We have engineered this strain to produce phenylalanine, which can then be used as a raw material for the synthesis of styrene via trans-cinnamic acid. To understand the response of this strain to the bioproducts of interest, we have analyzed the in-depth physiological and genetic response of the strain to these compounds. We found that in response to the exposure to the toxic compounds that the strain can produce, the cell launches a multifactorial response to enhance membrane impermeabilization. This process occurs via the activation of a cis to trans isomerase that converts cis unsaturated fatty acids to their corresponding trans isomers. In addition, the bacterial cells initiate a stress response program that involves the synthesis of a number of chaperones and ROS removing enzymes, such as peroxidases and superoxide dismutases. The strain also responds by enhancing the metabolism of glucose through the specific induction of the glucose phosphorylative pathway, Entner-Doudoroff enzymes, Krebs cycle enzymes and Nuo. In step with these changes, the cells induce two efflux pumps to extrude the toxic chemicals. Through analyzing a wide collection of efflux pump mutants, we found that the most relevant pump is TtgGHI, which is controlled by the TtgV regulator.

Stress-dependent macromolecular crowding in the mitochondrial matrix.

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.

Regulation of Cx43 and its role in trichloroethylene-induced cardiac toxicity in H9C2 rat cardiomyocytes.

Trichloroethylene (TCE), a widespread environmental contaminant, has been linked to congenital heart defects. Abnormal regulation of Connexin 43 is closely associated with various cardiac diseases. However, it is yet to be established how Cx43 responds to environmental pollutants. Here, we aim to explore the role of Cx43 in TCE-induced cardiac toxicity using H9C2 cardiomyocytes. EdU incorporation assay and cell cycle analysis revealed that increased number of TCE-treated cells entered into the S stage, indicating that TCE exposure provoked cell proliferation. Additionally, compromised mitochondrial function was observed in TCE-treated cells, and inhibition of mitochondrial permeability transition pore (mPTP) with Cyclosporin A or eliminating mitochondrial ROS by MitoQ alleviated the TCE-induced cardiac toxicity. Importantly, TCE exposure increased the protein expression levels of Cx43 and stimulated the recruitment of Cx43 to the mitochondria. TCE exposure disrupted canonical Wnt signal pathway, resulting in downregulation of antioxidant genes and ß-catenin. The adverse effects of TCE on Wnt signal pathway activation, mitochondrial function and cell proliferation were efficiently counteracted by either Cx43 knockdown or pharmaceutical activator of Wnt signaling, CHIR-99021. Taken together, our results for the first time revealed that dysregulation of Cx43 mediates TCE-induced heart defects via mitochondrial dysfunction and Wnt signaling inhibition, suggesting that Cx43 can be a potential molecular marker or therapeutic target for cardiac diseases caused by environmental pollutants.

Current progress on engineering microbial strains and consortia for production of cellulosic butanol through consolidated bioprocessing.

In the last decades, fermentative production of n-butanol has regained substantial interest mainly owing to its use as drop-in-fuel. The use of lignocellulose as an alternative to traditional acetone-butanol-ethanol fermentation feedstocks (starchy biomass and molasses) can significantly increase the economic competitiveness of biobutanol over production from non-renewable sources (petroleum). However, the low cost of lignocellulose is offset by its high recalcitrance to biodegradation which generally requires chemical-physical pre-treatment and multiple bioreactor-based processes. The development of consolidated processing (i.e., single-pot fermentation) can dramatically reduce lignocellulose fermentation costs and promote its industrial application. Here, strategies for developing microbial strains and consortia that feature both efficient (hemi)cellulose depolymerization and butanol production will be depicted, that is, rational metabolic engineering of native (hemi)cellulolytic or native butanol-producing or other suitable microorganisms; protoplast fusion of (hemi)cellulolytic and butanol-producing strains; and co-culture of (hemi)cellulolytic and butanol-producing microbes. Irrespective of the fermentation feedstock, biobutanol production is inherently limited by the severe toxicity of this solvent that challenges process economic viability. Hence, an overview of strategies for developing butanol hypertolerant strains will be provided.