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Metabolic Engineering Jun 2024Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks,...
Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used C-metabolic flux analysis (C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data was used to guide C-MFA model construction and to validate the C-MFA results. The C-MFA data was then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from β-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.
PubMed: 38942196
DOI: 10.1016/j.ymben.2024.06.010 -
Biotechnology For Biofuels and... Jun 2024Erythritol, a natural polyol, is a low-calorie sweetener synthesized by a number of microorganisms, such as Moniliella pollinis. Yet, a widespread use of erythritol is...
BACKGROUND
Erythritol, a natural polyol, is a low-calorie sweetener synthesized by a number of microorganisms, such as Moniliella pollinis. Yet, a widespread use of erythritol is limited by high production costs due to the need for cultivation on glucose-rich substrates. This study explores the potential of using Trichoderma reesei as an alternative host for erythritol production, as this saprotrophic fungus can be cultivated on lignocellulosic biomass residues. The objective of this study was to evaluate whether such an alternative host would lead to a more sustainable and economically viable production of erythritol by identifying suitable carbon sources for erythritol biosynthesis, the main parameters influencing erythritol biosynthesis and evaluating the feasibility of scaling up the defined process.
RESULTS
Our investigation revealed that T. reesei can synthesize erythritol from glucose but not from other carbon sources like xylose and lactose. T. reesei is able to consume erythritol, but it does not in the presence of glucose. Among nitrogen sources, urea and yeast extract were more effective than ammonium and nitrate. A significant impact on erythritol synthesis was observed with variations in pH and temperature. Despite successful shake flask experiments, the transition to bioreactors faced challenges, indicating a need for further scale-up optimization.
CONCLUSIONS
While T. reesei shows potential for erythritol production, reaching a maximum concentration of 1 g/L over an extended period, its productivity could be improved by optimizing the parameters that affect erythritol production. In any case, this research contributes valuable insights into the polyol metabolism of T. reesei, offering potential implications for future research on glycerol or mannitol production. Moreover, it suggests a potential metabolic association between erythritol production and glycolysis over the pentose phosphate pathway.
PubMed: 38937852
DOI: 10.1186/s13068-024-02537-x -
Langmuir : the ACS Journal of Surfaces... Jun 2024The emulsifying mechanism of supramolecular stereoisomeric sugar fatty acyl molecular gelators was evaluated. In-house-synthesized mannitol dioctanoate (M8) and sorbitol...
The emulsifying mechanism of supramolecular stereoisomeric sugar fatty acyl molecular gelators was evaluated. In-house-synthesized mannitol dioctanoate (M8) and sorbitol dioctanoate (S8) were tested. The stereoisomeric difference between the sugar groups significantly affected the gelation and emulsifying properties of the gelators. M8 and S8 formed oleogels at 2 and 3.5% (w/v) and emulsified water up to 30 and 60% (v/v), respectively. Microscopy showed that the gelator fibers are at the W/O interfaces, demonstrating a solid particle or network mode of stabilization. The long fibers of M8 were unable to completely encompass the water droplets, resulting in poor emulsification. Small, hair-like fibers of S8 showed better emulsification. When sunflower wax (SFW, 1% w/v) was added as a coemulsifier, synergetic action between the wax and S8 improved the stability of emulsions. Such synergy was not seen between SFW and M8, henceforth emulsion stability was not improved. This study proved that a subtle stereoisomeric difference at the molecular level can greatly alter the supramolecular and emulsifying properties of sugar-fatty acyl compounds.
PubMed: 38937253
DOI: 10.1021/acs.langmuir.3c03274 -
Viruses Jun 2024The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral...
The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.
Topics: Freeze Drying; SARS-CoV-2; COVID-19 Vaccines; Spike Glycoprotein, Coronavirus; Cryoprotective Agents; Trehalose; COVID-19; Animals; Humans; Mannitol; Sucrose; Vero Cells; Chlorocebus aethiops; Sorbitol; Drug Stability; Histidine; Vesicular stomatitis Indiana virus; Vaccines, Synthetic
PubMed: 38932234
DOI: 10.3390/v16060942 -
Pharmaceutics Jun 2024The present study compared vacuum drum drying (VDD) and conventional spray drying (SD) for solidifying crystalline ABT-199 nanosuspensions into redispersible oral drug...
The present study compared vacuum drum drying (VDD) and conventional spray drying (SD) for solidifying crystalline ABT-199 nanosuspensions into redispersible oral drug products. The aim was to optimize formulation compositions and process conditions to maintain nanoparticle size after tablet redispersion. The impact of drug load (22%, 33%, 44%) and type of drying protectant (mannitol, mannitol/trehalose mix (1:1), trehalose) on redispersibility and material powder properties were investigated. Moreover, compression analysis was performed assessing the influence of compaction pressure on primary nanocrystal redispersibility and tablet disintegration. Higher drug loads and lower drying protectant levels resulted in particle growth, confirming a drug load dependence on redispersibility behavior. Notably, all drying protectants showed similar protection properties at properly chosen drying process parameters (-dependent), except when VDD was used for mannitol formulations. Differences between the applied drying processes were observed in terms of downstream processing and tabletability: mannitol-containing formulations solidified via VDD showed an improved processability compared to formulations with trehalose. In conclusion, VDD is a promising drying technique that offers advantageous downstream processability compared to SD and represents an attractive novel processing technology for the pharmaceutical industry. As demonstrated in the present study, VDD combines higher yields with a leaner manufacturing process flow. The improved bulk properties provide enhanced tabletability and enable direct compression.
PubMed: 38931903
DOI: 10.3390/pharmaceutics16060782 -
Pharmaceutics May 2024Most infectious diseases of the gastrointestinal tract can easily be treated by exploiting the already available antibiotics with the change in administration approach...
Most infectious diseases of the gastrointestinal tract can easily be treated by exploiting the already available antibiotics with the change in administration approach and delivery system. Ciprofloxacin (CIP) is used as a drug of choice for many bacterial infections; however, long-term therapy and off-site drug accumulation lead to an increased risk of tendinitis and peripheral neuropathy. To overcome this issue, nanotechnology is being exploited to encapsulate antibiotics within polymeric structures, which not only facilitates dose maintenance at the infection site but also limits off-site side effects. Here, sodium alginate (SA) and thiol-anchored chitosan (TC) were used to encapsulate CIP via a calcium chloride (CaCl) cross-linker. For this purpose, the B-390 encapsulator was employed in the preparation of nanobeads using a simple technique. The hydrogel-like sample was then freeze-dried, using trehalose or mannitol as a lyoprotectant, to obtain a fine dry powder. Design of Experiment (DoE) was utilized to optimize the nanobead production, in which the influence of different independent variables was studied for their outcome on the polydispersity index (PDI), particle size, zeta potential, and percentage encapsulation efficiency (% EE). In vitro dissolution studies were performed in simulated saliva fluid, simulated gastric fluid, and simulated intestinal fluid. Antibacterial and anti-inflammatory studies were also performed along with cytotoxicity profiling. By and large, the study presented positive outcomes, proving the advantage of using nanotechnology in fabricating new delivery approaches using already available antibiotics.
PubMed: 38931815
DOI: 10.3390/pharmaceutics16060691 -
Nutrients Jun 2024Stroke is the world's second-leading cause of death. Current treatments for cerebral edema following intracerebral hemorrhage (ICH) mainly involve hyperosmolar fluids,...
Stroke is the world's second-leading cause of death. Current treatments for cerebral edema following intracerebral hemorrhage (ICH) mainly involve hyperosmolar fluids, but this approach is often inadequate. Propolis, known for its various beneficial properties, especially antioxidant and anti-inflammatory properties, could potentially act as an adjunctive therapy and help alleviate stroke-associated injuries. The chemical composition of propolis extract was analyzed by GC-MS after derivatization for its total phenolic and total flavonoid content. The total phenolic content and total flavonoid content of the propolis extract were 1037.31 ± 24.10 μg GAE/mL and 374.02 ± 3.36 μg QE/mL, respectively. By GC-MS analysis, its major constituents were found to be triterpenoids (22.4% of TIC). Minor compounds, such as phenolic lipids (6.7% of TIC, GC-MS) and diterpenic acids (2.3% of TIC, GC-MS), were also found. Ninety-six Sprague Dawley rats were divided into six groups; namely, the control group, the ICH group, and four ICH groups that received the following therapies: mannitol, propolis extract (daily oral propolis administration after the ICH induction), propolis-M (propolis and mannitol), and propolis-B+A (daily oral propolis administration 7 days prior to and 72 h after the ICH induction). Neurocognitive functions of the rats were analyzed using the rotarod challenge and Morris water maze. In addition, the expression of NF-κB, SUR1-TRPM4, MMP-9, and Aquaporin-4 was analyzed using immunohistochemical methods. A TUNEL assay was used to assess the percentage of apoptotic cells. Mannitol significantly improved cognitive-motor functions in the ICH group, evidenced by improved rotarod and Morris water maze completion times, and lowered SUR-1 and Aquaporin-4 levels. It also significantly decreased cerebral edema by day 3. Similarly, propolis treatments (propolis-A and propolis-B+A) showed comparable improvements in these tests and reduced edema. Moreover, combining propolis with mannitol (propolis-M) further enhanced these effects, particularly in reducing edema and the Virchow-Robin space. These findings highlight the potential of propolis from the Indonesian stingless bee, , from the Central Tapanuli region as a neuroprotective, adjunctive therapy.
Topics: Animals; Propolis; Neuroprotective Agents; Cerebral Hemorrhage; Rats, Sprague-Dawley; Bees; Disease Models, Animal; Rats; Male; Flavonoids; Antioxidants; Brain Edema; Gas Chromatography-Mass Spectrometry; Phenols
PubMed: 38931235
DOI: 10.3390/nu16121880 -
Plants (Basel, Switzerland) Jun 2024As a type of cell-wall-relaxing protein that is widely present in plants, expansins have been shown to actively participate in the regulation of plant growth and...
As a type of cell-wall-relaxing protein that is widely present in plants, expansins have been shown to actively participate in the regulation of plant growth and responses to environmental stress. Wild soybeans have long existed in the wild environment and possess abundant resistance gene resources, which hold significant value for the improvement of cultivated soybean germplasm. In our previous study, we found that the wild soybean expansin gene is specifically transcribed in roots, and its transcription level significantly increases under salt and drought stress. To further identify the function of , in this study, we cloned the CDS sequence of this gene. The transcription pattern of in the roots of wild soybean under salt and drought stress was analyzed by qRT-PCR. Using an -mediated genetic transformation, we obtained soybean hairy roots overexpressing . Under 150 mM NaCl- and 100 mM mannitol-simulated drought stress, the relative growth values of the number, length, and weight of transgenic soybean hairy roots were significantly higher than those of the control group. We obtained the transcriptomes of transgenic and wild-type soybean hairy roots under normal growth conditions and under salt and drought stress through RNA sequencing. A transcriptomic analysis showed that the transcription of genes encoding expansins (EXPB family), peroxidase, H-transporting ATPase, and other genes was significantly upregulated in transgenic hairy roots under salt stress. Under drought stress, the transcription of expansin (EXPB/LB family) genes increased in transgenic hairy roots. In addition, the transcription of genes encoding peroxidases, calcium/calmodulin-dependent protein kinases, and dehydration-responsive proteins increased significantly. The results of qRT-PCR also confirmed that the transcription pattern of the above genes was consistent with the transcriptome. The differences in the transcript levels of the above genes may be the potential reason for the strong tolerance of soybean hairy roots overexpressing the gene under salt and drought stress. In conclusion, the expansin can be used as a valuable candidate gene for the molecular breeding of soybeans.
PubMed: 38931088
DOI: 10.3390/plants13121656 -
Plants (Basel, Switzerland) Jun 2024Abiotic stresses pose a major increasing problem for the cultivation of maize. Autophagy plays a vital role in recycling and re-utilizing nutrients and adapting to...
Abiotic stresses pose a major increasing problem for the cultivation of maize. Autophagy plays a vital role in recycling and re-utilizing nutrients and adapting to stress. However, the role of autophagy in the response to abiotic stress in maize has not yet been investigated. Here, , which is essential for ATG8-PE conjugation, was isolated from the maize inbred line B73. The ATG3 sequence was conserved, including the C-terminal domains with HPC and FLKF motifs and the catalytic domain in different species. The promoter of the gene contained a number of elements involved in responses to environmental stresses or hormones. Heterologous expression of in yeast promoted the growth of strain under salt, mannitol, and low-nitrogen stress. The expression of could be altered by various types of abiotic stress (200 mM NaCl, 200 mM mannitol, low N) and exogenous hormones (500 µM ABA). GUS staining analysis of -GUS transgenic Arabidopsis revealed that gene activity increased after abiotic treatment. -overexpressing Arabidopsis plants had higher osmotic and salinity stress tolerance than wild-type plants. Overexpression of up-regulated the expression of other (, and ) under NaCl, mannitol and LN stress. These findings demonstrate that overexpression of can improve tolerance to multiple abiotic stresses.
PubMed: 38931070
DOI: 10.3390/plants13121637 -
Molecules (Basel, Switzerland) Jun 2024Acute lung injury (ALI) remains a significant global health issue, necessitating novel therapeutic interventions. In our latest study, we pioneered the use of...
Acute lung injury (ALI) remains a significant global health issue, necessitating novel therapeutic interventions. In our latest study, we pioneered the use of D-mannitol-cerium-quercetin/rutin coordination polymer nanoparticles (MCQ/R NPs) as a potential treatment for ALI. The MCQ/R NPs, which integrate rutin and quercetin for their therapeutic potential and D-mannitol for its pulmonary targeting, displayed exceptional efficacy. By utilizing cerium ions for optimal nanoparticle assembly, the MCQ/R NPs demonstrated an average size of less than 160 nm. Impressively, these nanoparticles outperformed conventional treatments in both antioxidative capabilities and biocompatibility. Moreover, our in vivo studies on LPS-induced ALI mice showed a significant reduction in lung tissue inflammation. This groundbreaking research presents MCQ/R NPs as a promising new approach in ALI therapeutics.
Topics: Acute Lung Injury; Quercetin; Animals; Mannitol; Nanoparticles; Mice; Polymers; Cerium; Rutin; Antioxidants; Humans; Drug Synergism; Disease Models, Animal; Lipopolysaccharides
PubMed: 38930884
DOI: 10.3390/molecules29122819