Phe-Gly motifs drive fibrillization of TDP-43's prion-like domain condensates.

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.

[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.

Slow Protein Dynamics Elicits New Enzymatic Functions by Means of Epistatic Interactions.

Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M ß-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried ß-sheet. The two mutations located in this ß-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M ß-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.

UPLC/ESI/MS profiling of red algae Galaxaura rugosa extracts and its activity against malaria mosquito vector, Anopheles pharoensis, with reference to Danio rerio and Daphnia magna as bioindicators.

BACKGROUND: Anopheles pharoensis has a major role in transmitting several human diseases, especially malaria, in Egypt?. Controlling Anopheles is considered as an effective strategy to eliminate the spread of malaria worldwide. Galaxaura rugosa is a species of red algae found in tropical to subtropical marine environments. The presence of G. rugosa is indicative of the ecosystem's overall health. The current work aims to investigate UPLC/ESI/MS profile of G. rugosa methanol and petroleum ether extracts and its activity against An. pharoensis and non-target organisms, Danio rerio and Daphnia magna. METHODS: Galaxaura rugosa specimens have been identified using DNA barcoding for the COI gene and verified as G. rugosa. The UPLC/ESI/MS profiling of G. rugosa collected from Egypt was described. The larvicidal and repellent activities of G. rugosa methanol and petroleum ether extracts against An. pharoensis were evaluated, as well as the toxicity of tested extracts on non-target organisms, Dan. rerio and Dap. magna. RESULTS: The UPLC/ESI/MS analysis of methanol and petroleum ether extracts led to the tentative identification of 57 compounds belonging to different phytochemical classes, including flavonoids, tannins, phenolic acids, phenyl propanoids. Larval mortality was recorded at 93.33% and 90.67% at 80 and 35 ppm of methanol and petroleum ether extracts, respectively, while pupal mortality recorded 44.44 and 22.48% at 35 and 30 ppm, respectively. Larval duration was recorded at 5.31 and 5.64 days by methanol and petroleum ether extracts at 80 and 35 ppm, respectively. A decrease in acetylcholinesterase (AChE) level and a promotion in Glutathione-S-transferase (GST) level of An. pharoensis 3rd instar larvae were recorded by tested extracts. The petroleum ether extract was more effective against An. pharoensis starved females than methanol extract. Also, tested extracts recorded LC50 of 1988.8, 1365.1, and 11.65, 14.36 µg/mL against Dan. rerio, and Dap. magna, respectively. CONCLUSIONS: Using red algae derivatives in An. pharoensis control could reduce costs and environmental impact and be harmless to humans and other non-target organisms.

Alleviating Chlorinated Alkane Inhibition on Dehalococcoides to Achieve Detoxification of Chlorinated Aliphatic Cocontaminants.

Cocontamination by multiple chlorinated solvents is a prevalent issue in groundwater, presenting a formidable challenge for effective remediation. Despite the recognition of this issue, a comprehensive assessment of microbial detoxification processes involving chloroethenes and associated cocontaminants, along with the underpinning microbiome, remains absent. Moreover, strategies to mitigate the inhibitory effects of cocontaminants have not been reported. Here, we revealed that chloroform exhibited the most potent inhibitory effects, followed by 1,1,1-trichloroethane and 1,1,2-trichloroethane, on dechlorination of dichloroethenes (DCEs) in Dehalococcoides-containing consortia. The observed inhibition could be attributed to suppression of biosynthesis and enzymatic activity of reductive dehalogenases and growth of Dehalococcoides. Notably, cocontaminants more profoundly inhibited Dehalococcoides populations harboring the vcrA gene than those possessing the tceA gene, thereby explaining the accumulation of vinyl chloride under cocontaminant stress. Nonetheless, we successfully ameliorated cocontaminant inhibition by augmentation with Desulfitobacterium sp. strain PR owing to its ability to attenuate cocontaminants, resulting in concurrent detoxification of DCEs, trichloroethanes, and chloroform. Microbial community analyses demonstrated obvious alterations in taxonomic composition, structure, and assembly of the dechlorinating microbiome in the presence of cocontaminants, and introduction of strain PR reshaped the dechlorinating microbiome to be similar to its original state in the absence of cocontaminants. Altogether, these findings contribute to developing bioremediation technologies to clean up challenging sites polluted with multiple chlorinated solvents.

Non-growth inhibitory doses of dimethyl sulfoxide alter gene expression and epigenetic pattern of bacteria.

Although dimethyl sulfoxide (DMSO) is a widely used solvent in scientific research, drug screening settings, and biomedical applications, its solvent (vehicle) effects on biological processes are overlooked. Using Escherichia coli as a model, we aimed to investigate and evaluate the effects of low-dose DMSO-driven changes in bacterial cells in a comprehensive and multifaceted manner by combining Fourier transform infrared spectroscopy analyses, analytical cell-biology approaches, and high-throughput sequencing. Here, we show that the non-toxic (1.0 and 2.5%, v/v) DMSO doses reduce the cellular levels of reactive oxygen species, change the cellular nucleic acid content and DNA topology, affect the global 5-methylcytosine pattern of the genome, and modulate gene transcription. These results indicate that even at non-toxic concentrations, DMSO is not inert: it can alter validity by changing or masking the assessed activity of the analyte. Besides, this manuscript does not only highlight that the low, non-toxic solvent doses of DMSO impinge on biological processes, including genome structure and function, but also, the high-throughput sequence data obtained during the study offer a platform for future research to elucidate the mechanism of epigenetically regulated genes in bacteria. KEY POINTS: • A clear-cut differentiation between the low-dose DMSO-treated and -untreated bacteria by PCA and LDA. • Drastic alterations in the DNA topology and nucleic acids of DMSO-treated bacteria. • Changes in transcriptome and epigenetic signatures with the low-dose DMSO.

On-column virus inactivation by solvent/detergent treatment for a recombinant biological product.

Each process step in the manufacture of biological products requires expensive resources and reduces total process productivity. Since downstream processing of biologicals is the main cost driver, process intensification is a persistent topic during the entire product life cycle. We present here one approach for the intensification of bioprocesses by applying on-column virus inactivation using solvent/detergent (S/D) treatment during ion-exchange chromatography. The established purification process of a recombinant protein was used as a model to compare key process parameters (i.e., product yield, specific activity, impurity clearance) of the novel approach to the standard process protocol. Additional wash and incubation steps with and without S/D-containing buffers were introduced to ensure sufficient contact time to effectively eliminate enveloped viruses and to significantly decrease the amount of S/D reagents. Comparison of key process parameters demonstrated equivalent process performance. To assess the viral clearance capacity of the novel approach, XMuLV was spiked as model virus to the chromatographic load and all resulting fractions were analyzed by TCID50 and RT-qPCR. Data indicates the inactivation capability of on-column virus inactivation even at 10% of the nominal S/D concentration, although the mechanism of viral clearance needs further investigation.

Cuscuta reflexa Possess Potent Inhibitory Activity Against Human Malaria Parasite: An In Vitro and In Vivo Study.

Drug resistance to practically all antimalarial drugs in use necessitate the development of new chemotherapeutics against malaria. In this aspect, traditionally used plants with folklore reputation are the pillar for drug discovery. Cuscuta reflexa being traditionally used in the treatment of malaria in Odisha, India we aimed to experimentally validate its antimalarial potential. Different solvent extracts of C. reflexa or column fractions from a promising solvent extract were evaluated for in vitro anti-plasmodial activity against Plasmodium falciparum strain Pf3D7. Potent fractions were further evaluated for inhibition of parasite growth against different drug resistant strains. Safety of these fractions was determined by in vitro cyto-toxicity, and therapeutic effectiveness was evaluated by suppression of parasitemia and improvement in survival of experimental mice. Besides, their immunomodulatory effect was investigated in Pf-antigen stimulated RAW cells. GCMS fingerprints of active fractions was determined. Column separation of methanol extract which showed the highest in vitro antiplasmodial activity (IC50 = 14.48 µg/ml) resulted in eleven fractions, three of which (F2, F3, and F4) had anti-plasmodial IC50 ranging from ≤ 10 to 2.2 µg/ml against various P. falciparum strains with no demonstration of in vitro cytotoxicity. F4 displayed the highest in vivo parasite suppression, and had a mean survival time similar to artesunate (19.3 vs. 20.6 days). These fractions significantly modulated expression of inflammatory cytokines in Pf-antigen stimulated RAW cells. The findings of the study confirm the antimalarial potential of C. reflexa. Exploration of phyto-molecules in GCMS fingerprints of active fractions is warranted for possible identification of lead anti-malarial phyto-drugs.

Phytochemicals Against Drug-Resistant Bacterial Biofilms and Use of Green Extraction Solvents to Increase Their Bioactivity.

Antimicrobial resistance has become one of the major global public health issues of the twenty-first century. One of the main factors in the limited action of antimicrobials is related to the ability of microorganisms, particularly bacteria, to form biofilms. These complex and well-organized communities allow the colonizing cells to acquire survival advantages over the same cells in suspension, including antibiotic resistance. A huge percentage of bacterial infections in humans are associated with biofilms, and many of them are chronic. Therefore, there is an urgent need to develop new products effective in controlling or eradicating biofilms. Plant secondary metabolites (phytochemicals) have demonstrated their potential as antibacterials against planktonic cells and sessile communities when used alone or in synergy with other molecules. This chapter covers recent advances in the activity of phytochemicals against biofilms, particularly those formed by drug-resistant bacteria. In addition, taking into account that the extraction step is crucial for the successful development of new bioactive compounds, the use of novel solvents that increase the phytochemical effect, such as natural deep eutectic solvents (NADES), as well as the recent applications of these solvents as antimicrobials are discussed.

Laser ablation of asphalt and coal in different solvents an In Vitro study.

Pulsed laser ablation in liquids (PLAL) is considered as green, cost effective, and facile method to produce nanocolloids which exhibit anticancer effect. When comparing breast cancer with other types of cancers, breast cancer is considered as the second cause of death in women. The objective of this article is to test the cytotoxicity of carbon-based materials prepared by PLAL on both the normal (REF) cell line and the human breast cancer (MCF7) cell line. In this study, PLAL is used to prepare nanocolloids of asphalt and coal in different solvents (ethanol, dimethyl sulfoxide (DMSO), phosphate buffer saline (PBS), and distilled water (DW)). A fiber laser of wavelength of 1.06 µm and an average power of 10 watts was used to prepare different nanocolloids in different solvents from asphalt and coal. The cytotoxic effect of the prepared materials was tested against breast cancer MCF7 cell line in vitro. The asphalt in both ethanol and DMSO was found to have a significant cytotoxic effect and the growth inhibition (GI) was found to be 62.1% and 50.5% at concentrations of 620 and 80 ppm respectively, unlike the coal in DMSO which showed G.I. of 59.5%. Both the prepared materials in the mentioned solvents showed low cytotoxicity against the normal cell line (REF). We can conclude that the organic materials prepared in organic solvents using the PLAL had shown a low cytotoxicity against the (REF) cell line while they exhibited a significant cytotoxic effect against the MCF7 cell line. Further studies are recommended to test these prepared materials in vivo.

Studying Fluorescence Sensing of Acetone and Tryptophan and Antibacterial Properties Based on Zinc-Based Triple Interpenetrating Metal-Organic Skeletons.

Two triple interpenetrating Zn(II)-based MOFs were studied in this paper. Named [Zn6(1,4-bpeb)4(IPA)6(H2O)]n (MOF-1) and {[Zn3(1,4-bpeb)1.5(DDBA)3]n·2DMF} (MOF-2), {1,4-bpeb = 1,4-bis [2-(4-pyridy1) ethenyl]benze, IPA = Isophthalic acid, DDBA = 3,3'-Azodibenzoic acid}, they were synthesized by the hydrothermal method and were characterized and stability tested. The results showed that MOF-1 had good acid-base stability and solvent stability. Furthermore, MOF-1 had excellent green fluorescence and with different phenomena in different solvents, which was almost completely quenched in acetone. Based on this phenomenon, an acetone sensing test was carried out, where the detection limit of acetone was calculated to be 0.00365% (volume ratio). Excitingly, the MOF-1 could also be used as a proportional fluorescent probe to specifically detect tryptophan, with a calculated detection limit of 34.84 µM. Furthermore, the mechanism was explained through energy transfer and competitive absorption (fluorescence resonance energy transfer (FRET)) and internal filtration effect (IFE). For antibacterial purposes, the minimum inhibitory concentrations of MOF-1 against Escherichia coli and Staphylococcus aureus were 19.52 µg/mL and 39.06 µg/mL, respectively, and the minimum inhibitory concentrations of MOF-2 against Escherichia coli and Staphylococcus aureus were 68.36 µg/mL and 136.72 µg/mL, respectively.

Insecticidal effects of some selected plant extracts against Anopheles stephensi (Culicidae: Diptera).

BACKGROUND: The use of synthetic insecticides against mosquitoes may lead to resistance development and potential health hazards in humans and the environment. Consequently, a paradigm needs to shift towards the alternative use of botanical insecticides that could strengthen an insecticide resistance management programme. This study aimed to assess the insecticidal effects aqueous, hexane, and methanol crude leaf extracts of Calpurnia aurea, Momordica foetida, and Zehneria scabra on an insectary colony of Anopheles stephensi larvae and adults. METHODS: Fresh leaves of C. aurea, M. foetida and Z. scabra were collected and dried, then separately ground to powder. Powdered leaves of test plants were extracted using sonication with aqueous, hexane, and methanol solvents. The extracts were concentrated, and a stock solution was prepared. For comparison, Temephos (Abate®) and control solutions (a mixture of water and emulsifier) were used as the positive and negative controls, respectively. Different test concentrations for the larvae and the adults were prepared and tested according to WHO (2005) and CDC (2010) guidelines to determine lethal concentration (LC) values. Mortality was observed after 24 h exposure. The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (Kruskal-Wallis test) and R software (a generalized linear model was used to determine LC50 and LC90 values of the extracts). RESULTS: The lowest LC50 values were observed in aqueous extracts of M. foetida followed by Z. scabra extract and C. aurea leaves at 34.61, 35.85, and 38.69 ppm, respectively, against the larvae. Larval mortality was not observed from the hexane extracts and negative control, while the standard larvicide (temephos) achieved 100% mortality. Further, the adulticidal efficacy was greatest for aqueous extract of Z. scabra with LC50 = 176.20 ppm followed by aqueous extract of C. aurea (LC50 = 297.75 ppm). CONCLUSION: The results suggest that the leaf extracts of the three test plants have the potential of being used for the control of vector An. stephensi larvae and adult instead of synthetic mosquitocides. Further studies need to be conducted to identify the active ingredients and their mode of action.

Modulating Activity Evaluation of Gut Microbiota with Versatile Toluquinol.

Gut microbiota have important implications for health by affecting the metabolism of diet and drugs. However, the specific microbial mediators and their mechanisms in modulating specific key intermediate metabolites from fungal origins still remain largely unclear. Toluquinol, as a key versatile precursor metabolite, is commonly distributed in many fungi, including Penicillium species and their strains for food production. The common 17 gut microbes were cultivated and fed with and without toluquinol. Metabolic analysis revealed that four strains, including the predominant Enterococcus species, could metabolize toluquinol and produce different metabolites. Chemical investigation on large-scale cultures led to isolation of four targeted metabolites and their structures were characterized with NMR, MS, and X-ray diffraction analysis, as four toluquinol derivatives (1-4) through O1/O4-acetyl and C5/C6-methylsulfonyl substitutions, respectively. The four metabolites were first synthesized in living organisms. Further experiments suggested that the rare methylsulfonyl groups in 3-4 were donated from solvent DMSO through Fenton's reaction. Metabolite 1 displayed the strongest inhibitory effect on cancer cells A549, A2780, and G401 with IC50 values at 0.224, 0.204, and 0.597 µM, respectively, while metabolite 3 displayed no effect. Our results suggest that the dominant Enterococcus species could modulate potential precursors of fungal origin and change their biological activity.

Influence of Two Widely Used Solvents, Ethanol and Dimethyl Sulfoxide, on Human Sperm Parameters.

To study mechanisms involved in fertility, many experimental assays are conducted by incubating spermatozoa in the presence of molecules dissolved in solvents such as ethanol (EtOH) or dimethyl sulfoxide (DMSO). Although a vehicle control group is usually included in such studies, it does not allow to evaluate the intrinsic effect of the solvent on sperm parameters and its potential influence on the outcome of the experiment. In the present study, we incubated human spermatozoa for 4 h in a capacitation medium in the absence or the presence of different concentrations of EtOH and DMSO (0.1, 0.5, 1.0, and 2.0%) to assess the impact of these solvents on sperm motility, vitality, capacitation, and acrosome integrity. The presence of statistically significant relationships between increasing solvent concentrations and the investigated parameters was assessed using linear mixed models. A significant effect was observed with both solvents for total and progressive sperm motilities. We also evaluated the effect of time for these parameters and showed that the influence of the solvents was stable between 0 and 4 h, indicating an almost direct impact of the solvents. While EtOH did not influence sperm vitality and acrosome integrity, a significant effect of increasing DMSO concentrations was observed for these parameters. Finally, regarding capacitation, measured via phosphotyrosine content, although a dose-dependent effect was observed with both solvents, the statistical analysis did not allow to precisely evaluate the intensity of the effect. Based on the results obtained in the present study, and the corresponding linear mixed models, we calculated the concentration of both solvents which would result in a 5% decline in sperm parameters. For EtOH, these concentrations are 0.9, 0.7, and 0.3% for total motility, progressive motility, and capacitation, respectively, while for DMSO they are 1.5, 1.1, >2, 0.3 and >2% for total motility, progressive motility, vitality, capacitation, and acrosome integrity, respectively. We recommend using solvent concentrations below these values to dissolve molecules used to study sperm function in vitro, to limit side effects.

Amauroderma rugosum Extract Suppresses Inflammatory Responses in Tumor Necrosis Factor Alpha/Interferon Gamma-Induced HaCaT Keratinocytes.

Keratinocytes form the physical barrier of the skin and play an important role in the inflammatory process. Amauroderma rugosum is an edible mushroom; however, its pharmacological properties have seldom been studied. Although the anti-inflammatory effect of the organic solvent extract of Amauroderma rugosum has been previously reported, it is not known whether the aqueous extract has a similar effect. In addition, the effect of Amauorderma rugosum extract on skin has never been explored. Therefore, the objectives of the present study were to evaluate the anti-inflammatory effects of the aqueous extract of Amauroderma rugosum on HaCaT keratinocytes, to explore its mechanisms of action, and to study the possible active ingredients involved. The results showed that the aqueous extract of Amauroderm rugosum at a concentration of 1.5 mg/mL was non-toxic to HaCaT cells and inhibited the release of cytokine interleukin-1ß, and chemokines interleukin-8 and monocyte chemoattractant protein-1 in tumor necrosis factor (TNF)-α- and interferon (IFN)-γ-stimulated HaCaT cells. Amauroderma rugosum extract reduced the intracellular levels of reactive oxygen species. In addition, Amauroderma rugosum extract reduced the total protein expression of nuclear factor-kappa B (NF-κB) and B-cells inhibitor alpha in HaCaT keratinocytes and inhibited the phosphorylation of mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (Akt), and mammalian target of rapamycin (mTOR) in TNF-α- and INF-γ-stimulated HaCaT keratinocytes. Chemical analysis revealed that the aqueous extract of Amauroderma rugosum contains polysaccharides, triterpenes, and phenolic compounds. Anti-inflammatory compounds, such as gallic acid, guanosine, and uridine, were also present. The anti-inflammatory effect of Amauroderma rugosum could be mimicked by a combination of gallic acid, guanosine, and uridine. In conclusion, our study suggests that the aqueous extract of Amauroderma rugosum exerts anti-inflammatory effects on keratinocytes through its antioxidant and inhibitory effects on MEK/ERK-, Akt/mTOR-, and NF-κB-dependent signaling pathways.

Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae through multi-objective medium optimization: aqueous two-phase purification and activity kinetics.

As physiological impairments that require replacement therapy continue to increase, so also does the need for improved production of acidic lipase from new microbial sources. Enterobacter cloacae strain UCCM 00116 produced a novel acidic lipase in kernel oil-processing waste-basal broth with 0.023:1 extracellular: intracellular localization ratio. This research re-directed enzyme localization to the extracellular milieu to reduce recovery cost using multi-objective response surface optimization of medium parameters. Results revealed a 1:0.32 extracellular:intracellular lipase ratio. Product formation kinetics, modeled by the Luedeking-Piret function, showed a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization through medium optimization. Aqueous two-phase system purification conditions extracted 95.22% lipase with 72.36 purity, a Vmax of 370.37 µmolmin-1, and a Km of 0.63 mmol. Enzyme activity was enhanced by K+ and Ca2+ ions, stable in many organic solvents, except acetone, and had pH and temperature optima at 2.5-3.5 and 50 °C, respectively.

Acute intrathecal administration of quipazine elicits air-stepping behavior.

Serotonin plays a pivotal role in the initiation and modulation of locomotor behavior in the intact animal, as well as following spinal cord injury. Quipazine, a serotonin 2 receptor agonist, has been used successfully to initiate and restore motor behavior in rodents. Although evidence suggests that the effects of quipazine are spinally mediated, it is unclear whether intrathecal (IT) quipazine administration alone is enough to activate locomotor-like activity or whether additional stimulation is needed. Thus, the current study examined the effects of IT administration of quipazine in postnatal day 1 rats in two separate experiments. In experiment 1, quipazine (0.1, 0.3, or 1.0 mg/kg) was dissolved in saline and administered via IT injection to the thoracolumbar cord. There was no significant effect of drug on hindlimb alternating stepping. In experiment 2, quipazine (0.3 or 1.0 mg/kg) was dissolved in a polysorbate 80-saline solution (Tween 80) and administered via IT injection. Polysorbate 80 was used to disrupt the blood-brain barrier to facilitate absorption of quipazine. The injection was followed by tail pinch 5 minutes post-injection. A significant increase in the percentage of hindlimb alternating steps was found in subjects treated with 0.3 mg/kg quipazine, suggesting that IT quipazine when combined with sensory stimulation to the spinal cord, facilitates locomotor-like behavior. These findings indicate that dissolving the drug in polysorbate 80 rather than saline may heighten the effects of IT quipazine. Collectively, this study provides clarification on the role of quipazine in evoking spinally-mediated locomotor behavior.

Solvent viscosity facilitates replication and ribozyme catalysis from an RNA duplex in a model prebiotic process.

The RNA World hypothesis posits that RNA was once responsible for genetic information storage and catalysis. However, a prebiotic mechanism has yet to be reported for the replication of duplex RNA that could have operated before the emergence of polymerase ribozymes. Previously, we showed that a viscous solvent enables information transfer from one strand of long RNA duplex templates, overcoming 'the strand inhibition problem'. Here, we demonstrate that the same approach allows simultaneous information transfer from both strands of long duplex templates. An additional challenge for the RNA World is that structured RNAs (like those with catalytic activity) function poorly as templates in model prebiotic RNA synthesis reactions, raising the question of how a single sequence could serve as both a catalyst and as a replication template. Here, we show that a viscous solvent also facilitates the transition of a newly synthesized hammerhead ribozyme sequence from its inactive, duplex state to its active, folded state. These results demonstrate how fluctuating environmental conditions can allow a ribozyme sequence to alternate between acting as a template for replication and functioning as a catalyst, and illustrate the potential for temporally changing environments to enable molecular processes necessary for the origin of life.

Effect of Three Defatting Solvents on the Techno-Functional Properties of an Edible Insect (Gryllus bimaculatus) Protein Concentrate.

Edible insects have received global attention as an alternative protein-rich food. However, their structural characteristics make them difficult to digest. To overcome this obstacle, we assessed the techno-functional properties of three protein concentrates from the cricket Gryllus bimaculatus. Freeze-dried G. bimaculatus powder was defatted using ethanol, hexene, or acetone as solvents, and the techno-functional properties (protein solubility, water and oil holding capacity, foaming properties, emulsion capacity, and gel formation) of the protein concentrates were determined. Freeze-dried G. bimaculatus powder comprised approximately 17.3% crude fat and 51.3% crude protein based on dry weight. Ethanol was the most effective solvent for reducing the fat content (from 17.30% to 0.73%) and increasing the protein content (from 51.3% to 62.5%) of the concentrate. Techno-functionality properties drastically differed according to the defatting solvent used and foaming properties were most affected. Thus, the techno-functional and whole properties must be considered for proper application of edible insects to achieve global food sustainability.

Segmented Flow Processes to Overcome Hurdles of Whole-Cell Biocatalysis in the Presence of Organic Solvents.

In modern process development, it is imperative to consider biocatalysis, and whole-cell catalysts often represent a favored form of such catalysts. However, the application of whole-cell catalysis in typical organic batch two-phase synthesis often struggles due to mass transfer limitations, emulsion formation, tedious work-up and, thus, low yields. Herein, we demonstrate that utilizing segmented flow tools enables the conduction of whole-cell biocatalysis efficiently in biphasic media. Exemplified for three different biotransformations, the power of such segmented flow processes is shown. For example, a 3-fold increase of conversion from 34 % to >99 % and a dramatic simplified work-up leading to a 1.5-fold higher yield from 44 % to 65 % compared to the analogous batch process was achieved in such a flow process.