Identifying Residues for Substrate Recognition in Human GPAT4 by Molecular Dynamics Simulations.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in triacylglycerol synthesis. Understanding its substrate recognition mechanism may help to design drugs to regulate the production of glycerol lipids in cells. In this work, we investigate how the native substrate, glycerol-3-phosphate (G3P), and palmitoyl-coenzyme A (CoA) bind to the human GPAT isoform GPAT4 via molecular dynamics simulations (MD). As no experimentally resolved GPAT4 structure is available, the AlphaFold model is employed to construct the GPAT4-substrate complex model. Using another isoform, GPAT1, we demonstrate that once the ligand binding is properly addressed, the AlphaFold complex model can deliver similar results to the experimentally resolved structure in MD simulations. Following the validated protocol of complex construction, we perform MD simulations using the GPAT4-substrate complex. Our simulations reveal that R427 is an important residue in recognizing G3P via a stable salt bridge, but its motion can bring the ligand to different binding hotspots on GPAT4. Such high flexibility can be attributed to the flexible region that exists only on GPAT4 and not on GPAT1. Our study reveals the substrate recognition mechanism of GPAT4 and hence paves the way towards designing GPAT4 inhibitors.

Glycerol Trinitrate Acts Downstream of Calcitonin Gene-Related Peptide in Trigeminal Nociception-Evidence from Rodent Experiments with Anti-CGRP Antibody Fremanezumab.

Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) have been recognized as important mediators in migraine but their mechanisms of action and interaction have not been fully elucidated. Monoclonal anti-CGRP antibodies like fremanezumab are successful preventives of frequent migraine and can be used to study CGRP actions in preclinical experiments. Fremanezumab (30 mg/kg) or an isotype control monoclonal antibody was subcutaneously injected to Wistar rats of both sexes. One to several days later, glyceroltrinitrate (GTN, 5 mg/kg) mimicking nitric oxide (NO) was intraperitoneally injected, either once or for three consecutive days. The trigeminal ganglia were removed to determine the concentration of CGRP using an enzyme-linked immunosorbent assay (ELISA). In one series of experiments, the animals were trained to reach an attractive sugar solution, the access to which could be limited by mechanical or thermal barriers. Using a semi-automated registration system, the frequency of approaches to the source, the residence time at the source, and the consumed solution were registered. The results were compared with previous data of rats not treated with GTN. The CGRP concentration in the trigeminal ganglia was generally higher in male rats and tended to be increased in animals treated once with GTN, whereas the CGRP concentration decreased after repetitive GTN treatment. No significant difference in CGRP concentration was observed between animals having received fremanezumab or the control antibody. Animals treated with GTN generally spent less time at the source and consumed less sugar solution. Without barriers, there was no significant difference between animals having received fremanezumab or the control antibody. Under mechanical barrier conditions, all behavioral parameters tended to be reduced but animals that had received fremanezumab tended to be more active, partly compensating for the depressive effect of GTN. In conclusion, GTN treatment seems to increase the production of CGRP in the trigeminal ganglion independently of the antibodies applied, but repetitive GTN administration may deplete CGRP stores. GTN treatment generally tends to suppress the animals' activity and increase facial sensitivity, which is partly compensated by fremanezumab through reduced CGRP signaling. If CGRP and NO signaling share the same pathway in sensitizing trigeminal afferents, GTN and NO may act downstream of CGRP to increase facial sensitivity.

Strategies for the synthesis of the osmolyte glucosylglycerate and its precursor glycerate.

Glycosidic osmolytes are widespread natural compounds that protect microorganisms and their macromolecules from the deleterious effects of various environmental stresses. Their protective properties have attracted considerable interest for industrial applications, especially as active ingredients in cosmetics and healthcare products. In that regard, the osmolyte glucosylglycerate is somewhat overlooked. Glucosylglycerate is typically accumulated by certain organisms when they are exposed to high salinity and nitrogen starvation, and its potent stabilizing effects have been demonstrated in vitro. However, the applications of this osmolyte have not been thoroughly explored due to the lack of a cost-efficient production process. Here, we present an overview of the progress that has been made in developing promising strategies for the synthesis of glucosylglycerate and its precursor glycerate, and discuss the remaining challenges. KEY POINTS: • Bacterial milking could be explored for fermentative production of glucosylglycerate • Glycoside phosphorylases of GH13_18 represent attractive alternatives for biocatalytic production • Conversion of glycerol with alditol oxidase is a promising strategy for generating the precursor glycerate.

Exploring the possible relationship between skin microbiome and brain cognitive functions: a pilot EEG study.

Human microbiota mainly resides on the skin and in the gut. Human gut microbiota can produce a variety of short chain fatty acids (SCFAs) that affect many physiological functions and most importantly modulate brain functions through the bidirectional gut-brain axis. Similarly, skin microorganisms also have identical metabolites of SCFAs reported to be involved in maintaining skin homeostasis. However, it remains unclear whether these SCFAs produced by skin bacteria can affect brain cognitive functions. In this study, we hypothesize that the brain's functional activities are associated with the skin bacterial population and examine the influence of local skin-bacterial growth on event-related potentials (ERPs) during an oddball task using EEG. Additionally, five machine learning (ML) methods were employed to discern the relationship between skin microbiota and cognitive functions. Twenty healthy subjects underwent three rounds of tests under different conditions-alcohol, glycerol, and water. Statistical tests confirmed a significant increase in bacterial population under water and glycerol conditions when compared to the alcohol condition. The metabolites of bacteria can turn phenol red from red-orange to yellow, confirming an increase in acidity. P3 amplitudes were significantly enhanced in response to only oddball stimulus at four channels (Fz, FCz, and Cz) and were observed after the removal of bacteria when compared with that under the water and glycerol manipulations. By using machine learning methods, we demonstrated that EEG features could be separated with a good accuracy (> 88%) after experimental manipulations. Our results suggest a relationship between skin microbiota and brain functions. We hope our findings motivate further study into the underlying mechanism. Ultimately, an understanding of the relationship between skin microbiota and brain functions can contribute to the treatment and intervention of diseases that link with this pathway.

Novel synergistic interactions between monolaurin, a mono-acyl glycerol and ß lactam antibiotics against Staphylococcus aureus: an in vitro study.

BACKGROUND: A major worldwide health issue is the rising frequency of resistance of bacteria.Drug combinations are a winning strategy in fighting resistant bacteria and might help in protecting the existing drugs.Monolaurin is natural compound extracted from coconut oil and has a promising antimicrobial activity against Staphylococcus.aureus. This study aims to examine the efficacy of monolaurin both individually and in combination with ß-lactam antibiotics against Staphylococcus aureus isolates. METHODS: Agar dilution method was used for determination of minimum inhibitory concentration (MIC) of monolaurin against S.aureus isolates. Scanning electron microscope (SEM) was used to detect morphological changes in S.aureus after treatment with monolaurin. Conventional and Real-time Polymerase chain reaction (RT-PCR) were performed to detect of beta-lactamase (blaZ) gene and its expressional levels after monolaurin treatment. Combination therapy of monolaurin and antibiotics was assessed through fractional inhibitory concentration and time-kill method. RESULTS: The antibacterial activity of monolaurin was assessed on 115 S.aureus isolates, the MIC of monolaurin were 250 to 2000 µg/ml. SEM showed cell elongation and swelling in the outer membrane of S.aureus in the prescence of 1xMIC of monolaurin. blaZ gene was found in 73.9% of S.aureus isolates. RT-PCR shows a significant decrease in of blaZ gene expression at 250 and 500 µg/ml of monolaurin. Synergistic effects were detected through FIC method and time killing curve. Combination therapy established a significant reduction on the MIC value. The collective findings from the antibiotic combinations with monolaurin indicated synergism rates ranging from 83.3% to 100%.In time-kill studies, combination of monolaurin and ß-lactam antibiotics produced a synergistic effect. CONCLUSION: This study showed that monolaurin may be a natural antibacterial agent against S. aureus, and may be an outstanding modulator of ß-lactam drugs. The concurrent application of monolaurin and ß-lactam antibiotics, exhibiting synergistic effects against S. aureus in vitro, holds promise as potential candidates for the development of combination therapies that target particularly, patients with bacterial infections that are nearly incurable.

Mitochondrial membrane transporters as attractive targets for the fermentative production of succinic acid from glycerol in Saccharomyces cerevisiae.

Previously, we reported an engineered Saccharomyces cerevisiae CEN.PK113-1A derivative able to produce succinic acid (SA) from glycerol with net CO2 fixation. Apart from an engineered glycerol utilization pathway that generates NADH, the strain was equipped with the NADH-dependent reductive branch of the TCA cycle (rTCA) and a heterologous SA exporter. However, the results indicated that a significant amount of carbon still entered the CO2-releasing oxidative TCA cycle. The current study aimed to tune down the flux through the oxidative TCA cycle by targeting the mitochondrial uptake of pyruvate and cytosolic intermediates of the rTCA pathway, as well as the succinate dehydrogenase complex. Thus, we tested the effects of deletions of MPC1, MPC3, OAC1, DIC1, SFC1, and SDH1 on SA production. The highest improvement was achieved by the combined deletion of MPC3 and SDH1. The respective strain produced up to 45.5 g/L of SA, reached a maximum SA yield of 0.66 gSA/gglycerol, and accumulated the lowest amounts of byproducts when cultivated in shake-flasks. Based on the obtained data, we consider a further reduction of mitochondrial import of pyruvate and rTCA intermediates highly attractive. Moreover, the approaches presented in the current study might also be valuable for improving SA production when sugars (instead of glycerol) are the source of carbon.

Insights into intraspecific diversity of central carbon metabolites in Saccharomyces cerevisiae during wine fermentation.

Saccharomyces cerevisiae is a major actor in winemaking that converts sugars from the grape must into ethanol and CO2 with outstanding efficiency. Primary metabolites produced during fermentation have a great importance in wine. While ethanol content contributes to the overall profile, other metabolites like glycerol, succinate, acetate or lactate also have significant impacts, even when present in lower concentrations. S. cerevisiae is known for its great genetic diversity that is related to its natural or technological environment. However, the variation range of metabolic diversity which can be exploited to enhance wine quality depends on the pathway considered. Our experiment assessed the diversity of primary metabolites production in a set of 51 S. cerevisiae strains from various genetic backgrounds. Results pointed out great yield differences depending on the metabolite considered, with ethanol having the lowest variation. A negative correlation between ethanol and glycerol was observed, confirming glycerol synthesis as a suitable lever to reduce ethanol yield. Genetic groups were linked to specific yields, such as the wine group and high α-ketoglutarate and low acetate yields. This research highlights the potential of using natural yeast diversity in winemaking. It also provides a detailed data set on production of well known (ethanol, glycerol, acetate) or little-known (lactate) primary metabolites.

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata.

BACKGROUND: Biofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach. RESULTS: Functional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements. CONCLUSIONS: The study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies.

Fluconazole-Loaded Ibuprofen In Situ Gel-Based Oral Spray for Oropharyngeal Candidiasis Treatment.

Oral candidiasis is a fungal infection affecting the oral mucous membrane, and this research specifically addresses on a localized treatment through fluconazole-loaded ibuprofen in situ gel-based oral spray. The low solubility of ibuprofen is advantageous for forming a gel when exposed to an aqueous phase. The 1% w/w fluconazole-loaded in situ gel oral sprays were developed utilizing various concentrations of ibuprofen in N-methyl pyrrolidone. The prepared solutions underwent evaluation for viscosity, surface tension, contact angle, water tolerance, gel formation, interface interaction, drug permeation, and antimicrobial studies. The higher amount of ibuprofen reduced the surface tension and retarded solvent exchange. The use of 50% ibuprofen as a gelling agent demonstrated prolonged drug permeation for up to 24 h. The incorporation of Cremophor EL in the formulations resulted in increased drug permeation and exhibited effective inhibition against Candida albicans, Candida krusei, Candida lusitaniae, and Candida tropicalis. While the Cremophor EL-loaded formulation did not exhibit enhanced antifungal effects on agar media, its ability to facilitate the permeation of fluconazole and ibuprofen suggested potential efficacy in countering Candida invasion in the oral mucosa. Moreover, these formulations demonstrated significant thermal inhibition of protein denaturation in egg albumin, indicating anti-inflammatory properties. Consequently, the fluconazole-loaded ibuprofen in situ gel-based oral spray presents itself as a promising dosage form for oropharyngeal candidiasis treatment.

Chemoenzymatic Synthesis of ABC-Type Enantiostructured Triacylglycerols by the Use of the p-Methoxybenzyl Protective Group.

This report demonstrates the first asymmetric synthesis of enantiopure structured triacylglycerols (TAGs) of the ABC type presenting three non-identical fatty acids, two of which are unsaturated. The unsaturated fatty acids included monounsaturated oleic acid (C18:1 n-9) and polyunsaturated linoleic acid (C18:2 n-6). This was accomplished by a six-step chemoenzymatic approach starting from (R)- and (S)-solketals. The highly regioselective immobilized Candida antarctica lipase (CAL-B) played a crucial role in the regiocontrol of the synthesis. The synthesis also benefited from the use of the p-methoxybenzyl (PMB) ether protective group, which enabled the incorporation of two different unsaturated fatty acids into the glycerol skeleton. The total of six such TAGs were prepared, four constituting the unsaturated fatty acids in the sn-1 and sn-2 positions, with a saturated fatty acid in the remaining sn-3 position of the glycerol backbone. In the two remaining TAGs, the different unsaturated fatty acids accommodated the sn-1 and sn-3 end positions, with the saturated fatty acid present in the sn-2 position. Enantiopure TAGs are urgently demanded as standards for the enantiospecific analysis of intact TAGs in fats and oils.

Peiella sedimenti gen. nov., sp. nov., a novel taxon within the family Caulobacteraceae isolated from sediment of a river.

A Gram-stain-negative bacterium, designated XZ-24T, was isolated from sediment of a river in Mianyang city, Sichuan province, PR China. Cells (1.0-2.0 µm long and 0.4-0.5 µm in width) were strictly aerobic, non-spore-forming, rod shaped, prosthecate and motile by means of a polar flagellum. Growth occurred at 10-37 °C (optimum, 30 °C), at pH 5.0-9.0 (optimum pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum 1.0 % NaCl). The results of phylogenetic analysis based on genomes and 16S rRNA gene sequences indicated that XZ-24T formed a distinct phyletic branch within the family Caulobacteraceae and was most closely related to members of the genera Brevundimonas, Caulobacter and Phenylobacterium with 95.3-96.5 % 16S rRNA gene sequence similarities. The average amino acid identities (AAI) between XZ-24T and species of the family Caulobacteraceae were 47.0-64.5 %, which were below the genus boundary (70 %). The predominant cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, C18 : 1ω7c 11-methyl and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), the isoprenoid quinone was Q-10, and the major polar lipids were 1,2-di-O-acyl-3-O-α-d-glucopyranuronosyl glycerol; 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d glucopyranuronosyl] glycerol and phosphatidylglycerol. The genome size of XZ-24T was 2.64 Mb with a DNA G+C content of 68.9 %. On the basis of the evidence presented in this study, strain XZ-24T represents a novel species of a novel genus in the family Caulobacteraceae, for which the name Peiella sedimenti gen. nov., sp. nov. (Type strain XZ-24T=CCTCC AB 20 23 094T=KCTC 8038T) is proposed.

How Should Regulators and Manufacturers Prevent Avoidable Deaths of Children From Contaminated Cough Syrup?

This commentary responds to a case about diethylene glycol-contaminated glycerin in cough syrup. Glycerin is a commonly used excipient in medicines to improve texture and taste. Excipients are typically pharmacologically inactive ingredients contained in prescription and over-the-counter drugs that play a critical role in the delivery, effectiveness, and stability of active drug substances. The commentary first canvasses how contaminants enter the excipient supply chains. One way is by misleading labeling or intentional adulteration by manufacturers or suppliers. Another way is by human or systemic error. This commentary then discusses quality control testing and suggests the ethical and clinical importance of increased transparency in excipient supply chains.

Comparing methanol and glycerol as carbon sources for mainstream partial denitrification/anammox in an IFAS process.

This study explored the implementation of mainstream partial denitrification with anammox (PdNA) in the second anoxic zone of a wastewater treatment process in an integrated fixed film activated sludge (IFAS) configuration. A pilot study was conducted to compare the use of methanol and glycerol as external carbon sources for an IFAS PdNA startup, with a goal to optimize nitrogen removal while minimizing carbon usage. The study also investigated the establishment of anammox bacteria on virgin carriers in IFAS reactors without the use of seeding, and it is the first IFAS PdNA startup to use methanol as an external carbon source. The establishment of anammox bacteria was confirmed in both reactors 102 days after startup. Although the glycerol-fed reactor achieved a higher steady-state maximum ammonia removal rate because of anammox bacteria (1.6 ± 0.3 g/m2/day) in comparison with the methanol-fed reactor (1.2 ± 0.2 g/m2/day), both the glycerol- and methanol-fed reactors achieved similar average in situ ammonia removal rates of 0.39 ± 0.2 g/m2/day and 0.40 ± 0.2 g/m2/day, respectively. Additionally, when the upstream ammonia versus NOx (AvN) control system maintained an ideal ratio of 0.40-0.50 g/g, the methanol-fed reactor attained a lower average effluent TIN concentration (3.50 ± 1.2 mg/L) than the glycerol-fed reactor (4.43 ± 1.6 mg/L), which was prone to elevated nitrite concentrations in the effluent. Overall, this research highlights the potential for PdNA in IFAS configurations as an efficient and cost-saving method for wastewater treatment, with methanol as a viable carbon source for the establishment of anammox bacteria. PRACTITIONER POINTS: Methanol is an effective external carbon source for an anammox startup that avoids the need for costly alternative carbon sources. The methanol-fed reactor demonstrated higher TIN removal compared with the glycerol-fed reactor because of less overproduction of nitrite. Anammox bacteria was established in an IFAS reactor without seeding and used internally stored carbon to reduce external carbon addition. Controlling the influent ammonia versus NOx (AvN) ratio between 0.40 and 0.50 g/g allowed for low and stable TIN effluent conditions.

Comprehensive molecular-isotopic characterization of archaeal lipids in the Black Sea water column and underlying sediments.

The Black Sea is a permanently anoxic, marine basin serving as model system for the deposition of organic-rich sediments in a highly stratified ocean. In such systems, archaeal lipids are widely used as paleoceanographic and biogeochemical proxies; however, the diverse planktonic and benthic sources as well as their potentially distinct diagenetic fate may complicate their application. To track the flux of archaeal lipids and to constrain their sources and turnover, we quantitatively examined the distributions and stable carbon isotopic compositions (δ13 C) of intact polar lipids (IPLs) and core lipids (CLs) from the upper oxic water column into the underlying sediments, reaching deposits from the last glacial. The distribution of IPLs responded more sensitively to the geochemical zonation than the CLs, with the latter being governed by the deposition from the chemocline. The isotopic composition of archaeal lipids indicates CLs and IPLs in the deep anoxic water column have negligible influence on the sedimentary pool. Archaeol substitutes tetraether lipids as the most abundant IPL in the deep anoxic water column and the lacustrine methanic zone. Its elevated IPL/CL ratios and negative δ13 C values indicate active methane metabolism. Sedimentary CL- and IPL-crenarchaeol were exclusively derived from the water column, as indicated by non-variable δ13 C values that are identical to those in the chemocline and by the low BIT (branched isoprenoid tetraether index). By contrast, in situ production accounts on average for 22% of the sedimentary IPL-GDGT-0 (glycerol dibiphytanyl glycerol tetraether) based on isotopic mass balance using the fermentation product lactate as an endmember for the dissolved substrate pool. Despite the structural similarity, glycosidic crenarchaeol appears to be more recalcitrant in comparison to its non-cycloalkylated counterpart GDGT-0, as indicated by its consistently higher IPL/CL ratio in sediments. The higher TEX86 , CCaT, and GDGT-2/-3 values in glacial sediments could plausibly result from selective turnover of archaeal lipids and/or an archaeal ecology shift during the transition from the glacial lacustrine to the Holocene marine setting. Our in-depth molecular-isotopic examination of archaeal core and intact polar lipids provided new constraints on the sources and fate of archaeal lipids and their applicability in paleoceanographic and biogeochemical studies.

An improved mouse model of sepsis based on intraperitoneal injections of the enriched culture of cecum slurry.

AIM: Sepsis is a life-threatening clinical syndrome comprising multiorgan dysfunctions caused by a disproportionate body immune response. There are several animal sepsis models which are based on cecum ligation, cecal puncture, and cecum slurry injection. The major limitation of all current sepsis models is the high variability owing to the variable degree of ligation, puncture and inconsistent microbial composition used for sepsis initiation. The primary objective of this work is to demonstrate the feasibility of a standardized method for sepsis development. MATERIALS AND METHODS: The cecal slurry bacterial culture was developed and preserved in glycerol stocks. Antibiotics aztreonam and vancomycin were used for generating several defined, enriched cecal slurry bacterial cultures. Mice survival was assessed until 48 hrs post injection, and the tissue samples were collected after 10 hrs from sepsis initiation. KEY FINDINGS: The results indicate that increasing polymicrobial load resulted in lower survival rates and was associated with the higher number of infiltrating immune cells and necrosis. H&E (haematoxylin & eosin) staining & serum markers revealed that septic mice exhibited increased inflammation and significant damage to the liver and kidneys. The defined Gram-negative and Gram-positive specific cecal slurry bacterial cultures were developed and their efficiency in inducing sepsis was characterized. SIGNIFICANCE: Enriched cecal slurry bacterial cultures can be stored in glycerol stocks at -80 °C. This has an ethical advantage of avoiding unnecessary animal euthanasia for each experiment and provides a standardization capability of sepsis development.

Hormetic effects of a cannabinoid system component, 2-arachidonoyl glycerol, on cell viability and expression profile of growth factors in cultured mouse Sertoli cells: Friend or foe of male fertility?

The generally undesired effects of exocannabinoids on male reproduction include alterations in testicular cell proliferation and function, as well as apoptosis induction. However, this paradigm has been challenged by the ability of endocannabinoids to regulate reproductive function. The present study addresses these paradoxical facts by investigating the effects of the endocannabinoid 2-arachidonoyl glycerol (2-AG) on mouse Sertoli cells' survival and apoptosis, with a mechanistic insight into Sertoli cell-based growth factors' production. The Mus musculus Sertoli cell line (TM4) was exposed to different concentrations of 2-AG, and cell viability was evaluated using MTT assay. Growth factors' gene and protein expressions were analyzed through RT-PCR and western blotting. 2-AG concentration dependently increased TM4 viability, with a slight increase starting at 0.0001 µM, a peak of 190% of the control level at 1 µM, and a decrease at 3 µM. Moreover, 2-AG paradoxically altered mRNA expression of caspase-3 and growth factors. Caspase-3 mRNA expression was down-regulated, and growth factors mRNA and protein expression were up-regulated when using a low concentration of 2-AG (1 µM). Opposite effects were observed by a higher concentration of 2-AG (3 µM). These paradoxical effects of 2-AG can be explained through the concept of hormesis. The results indicate the pivotal role of 2-AG in mediating Sertoli cell viability and apoptosis, at least in part, through altering growth factors secretion. Furthermore, they suggest the involvement of endocannabinoids in Sertoli cell-based physiological and pathological conditions and reflect the ability of abnormally elevated 2-AG to mimic the actions of exocannabinoids in reproductive dysfunction.

[Enhancing the glycerol utilization of engineered yeast increases its bisabolene production].

Bisabolene is a compound commonly found in essential oils of various plants. It has a broad application in sectors such as chemical, pharmaceutical, and health-care products. This study focuses on modifying the glycerol metabolism pathway to obtain a high bisabolene-producing strain of Saccharomyces cerevisiae. To achieve this, the glycerol transporter gene PtFPS2 from Pachysolen tannophilus and the glycerol dehydrogenase gene Opgdh from Ogataea parapolymorpha were overexpressed in engineered yeast YS036, which was equipped with a GAL promoters-enhanced mevalonic acid pathway. Additionally, the glucose-inhibiting transcription factor MIG1 was knocked out to reduce glucose inhibition. The results showed that the GAL promoter transcription levels of the recombinant yeast strains increased, and the co-utilization of sucrose and glycerol was further improved in MIG1-knockout strain. Moreover, the maximum yield of bisabolene in shaking flask fermentation increased to 866.7 mg/L, an 82.2% increase compared to that of the original strain. By modifying the metabolic pathway of carbon sources, the yield of bisabolene was considerably improved. This study offers an effective strategy for enhancing the yield of terpene compounds in engineered yeast.

[Tolerance of hyperosmolar Candida krusei].

The utilization of industrial microorganisms for the conversion of lignocellulose into high value-added chemicals is an essential pathway towards achieving carbon neutrality and promoting sustainable bioeconomy. However, the pretreated lignocellulase hydrolysate often contains various sugars, salts, phenols/aldehydes and other substances, which requires microorganisms to possess strong tolerance for direct fermentation. This study aims to investigate the tolerance of Candida krusei to substrate, salt, and high temperature shock, in order to validate its potential for utilizing the enzymatic hydrolysate of Pennisetum giganteum in seawater for fermentation. The experimental results showed that the adaptively domesticated C. krusei exhibited tolerance to glucose at a concentration of 200 g/L and became a hypertonic strain. When seawater was used instead of freshwater without sterilization, the yield of glycerol in fermentation was 109% higher than that in freshwater with sterilization. Moreover, the combined thermal shock at 32 hours of fermentation and addition of 10 Na2SO3 at 48 hours resulted in a yield of glycerol to glucose 0.37 g/g, which was 225% higher than the control group. By fermenting the enzymatic hydrolysate of P. giganteum pretreated in seawater, the total conversion rate of glucose into glycerol and ethanol reached 0.45 g/g. This study indicates that hypertonic C. krusei exhibits remarkable adaptability to substrate, salt, and temperature. It not only can directly utilize complex lignocellulosic hydrolysates, but also exhibits strong tolerance to them. Therefore, it provides a potential candidate strain for the production of bio-based chemicals using lignocellulosic processes.

Peatland warming influences the abundance and distribution of branched tetraether lipids: Implications for temperature reconstruction.

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane lipids whose distribution in peatland soils serves as an important proxy for past climate changes due to strong linear correlations with temperature in modern environments. However, commonly used brGDGT-based temperature models are characterized by high uncertainty (ca. 4 °C) and these calibrations can show implausible correlations when applied at an ecosystem level. This lack of accuracy is often attributed to our limited understanding of the exact mechanisms behind the relationship between brGDGTs and temperature and the potential effect of temperature-independent factors on brGDGT distribution. Here, we examine the abundance and distribution of brGDGTs in a boreal peatland after four years of in-situ warming (+0, +2.25, +4.5, +6.75 and +9 °C). We observed that with warming, concentrations of total brGDGTs increased. Furthermore, we determined a shift in brGDGT distribution in the surface aerobic layers of the acrotelm (0-30 cm depth), whereas no detectable change was observed at deeper anaerobic depths (>40 cm), possibly due to limited microbial activity. The response of brGDGTs to warming was also reflected by a strong increase in the methylation index of 5-methyl brGDGTs (MBT'5Me), classically used as a temperature proxy. Further, the relationship between the MBT'5Me index and soil temperature differed between 0-10, 10-20 and 20-30 cm depth, highlighting depth-specific response of brGDGTs to warming, which should be considered in paleoenvironmental and paleoecological studies. As the bacterial community composition was generally unaltered, the rapid changes in brGDGT distribution argue for a physiological adaptation of the microorganisms producing these lipids. Finally, soil temperature and water table depth were better predictors of brGDGT concentration and distribution, highlighting the potential for these drivers to impact brGDGT-based proxies. To summarize, our results provide insights on the response of brGDGT source microorganisms to soil warming and underscore brGDGTs as viable temperature proxies for better understanding of climatic perturbation in peatlands.

Bringing a scalable adaptive hybrid modeling framework closer to industrial use: Application on a multiscale fungal fermentation.

Digitalization has paved the way for new paradigms such as digital shadows and digital twins for fermentation processes, opening the door for real-time process monitoring, control, and optimization. With a digital shadow, real-time model adaptation to accommodate complex metabolic phenomena such as metabolic shifts of a process can be monitored. Despite the many benefits of digitalization, the potential has not been fully reached in the industry. This study investigates the development of a digital shadow for a very complex fungal fermentation process in terms of microbial physiology and fermentation operation on pilot-scale at Novonesis and the challenges thereof. The process has historically been difficult to optimize and control due to a lack of offline measurements and an absence of biomass measurements. Pilot-scale and lab-scale fermentations were conducted for model development and validation. With all available pilot-scale data, a data-driven soft sensor was developed to estimate the main substrate concentration (glucose) with a normalized root mean squared error (N-RMSE) of 2%. This robust data-driven soft sensor was able to estimate accurately in lab-scale (volume < 20× pilot) with a N-RMSE of 7.8%. A hybrid soft sensor was developed by combining the data-driven soft sensor with a mass balance to estimate the glycerol and biomass concentrations on pilot-scale data with N-RMSEs of 11% and 21%, respectively. A digital shadow modeling framework was developed by coupling a mechanistic model (MM) with the hybrid soft sensor. The digital shadow modeling framework significantly improved the predictability compared with the MM. The contribution of this study brings the application of digital shadows closer to industrial implementation. It demonstrates the high potential of using this type of modeling framework for scale-up and leads the way to a new generation of in silico-based process development.