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Metabolic Engineering Jun 20247-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D. Despite significant improvements in the biosynthesis of...
7-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D. Despite significant improvements in the biosynthesis of 7-DHC, it remains insufficient to meet the industrial demands. In this study, we reported high-level production of 7-DHC in an industrial Saccharomyces cerevisiae leveraging subcellular organelles. Initially, the copy numbers of DHCR24 were increased in combination with sterol transcriptional factor engineering and rebalanced the redox power of the strain. Subsequently, the effects of compartmentalizing the post-squalene pathway in peroxisomes were validated by assembling various pathway modules in this organelle. Furthermore, several peroxisomes engineering was conducted to enhance the production of 7-DHC. Utilizing the peroxisome as a vessel for partial post-squalene pathways, the potential of yeast for 7-dehydrocholesterol production was demonstrated by achieving a 26-fold increase over the initial production level. 7-DHC titer reached 640.77 mg/L in shake flasks and 4.28 g/L in a 10 L bench-top fermentor, the highest titer ever reported. The present work lays solid foundation for large-scale and cost-effective production of 7-DHC for practical applications.
PubMed: 38936763
DOI: 10.1016/j.ymben.2024.06.011 -
Current Biology : CB Jun 2024Dinophysis dinoflagellates are predators of Mesodinium ciliates, from which they retain only the plastids of cryptophyte origin. The absence of nuclear photosynthetic...
Dinophysis dinoflagellates are predators of Mesodinium ciliates, from which they retain only the plastids of cryptophyte origin. The absence of nuclear photosynthetic cryptophyte genes in Dinophysis raises intriguing physiological and evolutionary questions regarding the functional dynamics of these temporary kleptoplastids within a foreign cellular environment. In an experimental setup including two light conditions, the comparative analysis with Mesodinium rubrum and the cryptophyte Teleaulax amphioxeia revealed that Dinophysis acuminata possessed a smaller and less dynamic functional photosynthetic antenna for green light, a function performed by phycoerythrin. We showed that the lack of the cryptophyte nucleus prevented the synthesis of the phycoerythrin α subunit, thereby hindering the formation of a complete phycoerythrin in Dinophysis. In particular, biochemical analyses showed that Dinophysis acuminata synthesized a poorly stable, incomplete phycoerythrin composed of chromophorylated β subunits, with impaired performance. We show that, consequently, a continuous supply of new plastids is crucial for growth and effective photoacclimation in this organism. Transcriptome analyses revealed that all examined strains of Dinophysis spp. have acquired the cryptophyte pebA and pebB genes through horizontal gene transfer, suggesting a potential ability to synthesize the phycobilin pigments bound to the cryptophyte phycoerythrin. By emphasizing that a potential long-term acquisition of the cryptophyte plastid relies on establishing genetic independence for essential functions such as light harvesting, this study highlights the intricate molecular challenges inherent in the enslavement of organelles and the processes involved in the diversification of photosynthetic organisms through endosymbiosis.
PubMed: 38936366
DOI: 10.1016/j.cub.2024.05.066 -
Current Opinion in Cell Biology Jun 2024Membrane remodelling is essential for the trafficking of macromolecules throughout the cell, a process that regulates various aspects of cellular health and pathology.... (Review)
Review
Membrane remodelling is essential for the trafficking of macromolecules throughout the cell, a process that regulates various aspects of cellular health and pathology. Recent studies implicate the role of biomolecular condensates in regulating multiple steps of the membrane trafficking pathway including but not limited to the organization of the trafficking machinery, dynamic remodeling of membranes, spatial and functional regulation, and response to cellular signals. The implicated proteins contain key structural elements, most notably prion-like domains within intrinsically disordered regions that are necessary for biomolecular condensate formation at fusion sites in processes like endocytic assembly, autophagy, organelle biosynthesis and synaptic vesicle fusion. Experimental and theoretical advances in the field continue to demonstrate that protein condensates can perform mechanical work, the implications of which can be extrapolated to diverse areas of membrane biology.
PubMed: 38936257
DOI: 10.1016/j.ceb.2024.102393 -
Journal of Phycology Jun 2024Mechanical damage to a cell can be fatal, and the cell must reseal its membrane and restore homeostasis to survive. Plant cell repair involves additional steps such as...
Mechanical damage to a cell can be fatal, and the cell must reseal its membrane and restore homeostasis to survive. Plant cell repair involves additional steps such as rebuilding vacuoles, rearranging chloroplasts, and remodeling the cell wall. When we pierced a Griffithsia monilis cell with a glass needle, a large amount of intracellular contents was released, but the cell membrane resealed in less than a second. The turgor of the vacuole was quickly restored, and the punctured cell returned to its original shape within an hour. Organelles such as chloroplasts and nuclei migrated to the wound site for 12 h and then dispersed throughout the cell after the wound was covered by a new cell wall. Using fluorescent probes, high levels of reactive oxygen species (ROS) and calcium were detected at the wound site from 3 h after wounding, which disappeared when cell repair was complete. Wounding in a solution containing ROS scavengers inhibited cellular repair, and inhibiting nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity or blocking calcium influx reversibly inhibited cell repair. Oryzalin reversibly inhibited both chloroplast movement and ROS production during cell repair. Our results show that cell repair in G. monilis is regulated by calcium-mediated ROS signaling and that microtubules serve as mechanical effectors.
PubMed: 38935837
DOI: 10.1111/jpy.13476 -
PloS One 2024Diet-induced obesity reduces oocyte quality mainly by impacting oocyte mitochondrial functions. Moreover, maternal obesity is associated with mitochondrial dysfunction...
Diet-induced obesity reduces oocyte quality mainly by impacting oocyte mitochondrial functions. Moreover, maternal obesity is associated with mitochondrial dysfunction in oocytes of their adult offspring. However, these effects were reported only in fully grown oocytes, mainly in the form of abnormal mitochondrial ultrastructure. It is unknown if obesogenic (OB) diets or maternal obesity already impact the primordial and preantral follicles. Considering the long duration and dynamics of folliculogenesis, determining the stage at which oocytes are affected and the extent of the damage is crucial for optimal reproductive management of obese patients and their daughters. Potential interaction between maternal and offspring diet effects are also not described, yet pivotal in our contemporary society. Therefore, here we examined the impact of OB diets on oocyte mitochondrial ultrastructure in primordial and activated preantral follicles in offspring from diet-induced obese or lean mothers. We used an outbred Swiss mouse model to increase the pathophysiological relevance to humans. Female mice were fed control or OB diets for 7 weeks, then mated with control males. Their female offspring were fed control or OB diets after weaning for 7 weeks (2-by-2 factorial design). Adult offspring ovarian sections were examined using transmission electron microscopy. We characterised and classified unique features of oocyte mitochondrial ultrastructure in the preantral follicles. An increase in mitochondrial matrix density was the most predominant change during follicle activation in secondary follicles, a feature that is linked with a higher mitochondrial activity. Maternal obesity increased mitochondrial density already in the primordial follicles suggesting an earlier increase in bioenergetic capacity. Maternal obesity did not induce abberant ultrastructure (abnormalities and defects) in primordial or preantral follicles. In contrast, offspring OB diet increased mitochondrial abnormalities in the primordial follicles. Further investigation of the consequences of these changes on oocyte metabolic regulation and stress levels during folliculogenesis is needed.
Topics: Animals; Oocytes; Female; Ovarian Follicle; Mice; Mitochondria; Pregnancy; Obesity; Male; Obesity, Maternal; Prenatal Exposure Delayed Effects; Diet, High-Fat
PubMed: 38935642
DOI: 10.1371/journal.pone.0305912 -
The Journal of Cell Biology Oct 2024Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists...
Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield ∼60 transformants/μg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism.
Topics: Aureobasidium; Cell Nucleus; Genetic Vectors; Green Fluorescent Proteins; Homologous Recombination; Luminescent Proteins; Red Fluorescent Protein; Transformation, Genetic; Genetic Techniques
PubMed: 38935076
DOI: 10.1083/jcb.202402114 -
Anatomia, Histologia, Embryologia Jul 2024The biobanks from dermal biopsies represent an interesting strategy for biodiversity conservation. Nevertheless, the morphological and cellular patterns of the dermis...
The biobanks from dermal biopsies represent an interesting strategy for biodiversity conservation. Nevertheless, the morphological and cellular patterns of the dermis can be influenced by the age and sex of the individual. Therefore, evaluating these factors is interesting for forming biobanks of Antillean manatees. These animals, representatives of marine fauna, have had their population reduced, and biobanks are essential for their conservation. Then, we evaluated the effects of age (3.5 years vs. 3.6-16 years vs. 23.6 years) and sex (males vs. females) on morphological and cellular parameters using histological and in vitro culture techniques. Regardless of age, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery. Nonetheless, fibroblast reduction was observed in groups aged 23.6 years compared to other animals (p < 0.05). Additionally, cells from animals aged 3.6-16 years showed more significant mitochondrial damage than the other groups (p < 0.05). Regardless of sex, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery; however, females had fewer fibroblasts than males (p < 0.05). Cells from females showed lower mitochondrial damage when compared to cells from males. In summary, although age and sex do not influence dermal thickness and cell recovery, variations in the number of fibroblasts and mitochondrial characteristics were observed among the groups. These differences may be significant for understanding the dermis aspects to be correlated to biobank systems.
Topics: Animals; Male; Female; Fibroblasts; Dermis; Trichechus manatus; Sex Factors; Age Factors; Collagen; Mitochondria; Cell Proliferation
PubMed: 38934868
DOI: 10.1111/ahe.13081 -
Molecular Biology and Evolution Jun 2024Plant cells harbor two membrane-bound organelles containing their own genetic material -plastids and mitochondria. Although the two organelles co-exist and co-evolve...
Plant cells harbor two membrane-bound organelles containing their own genetic material -plastids and mitochondria. Although the two organelles co-exist and co-evolve within the same plant cells, they differ in genome copy number, intracellular organization, and mode of segregation. How these attributes affect the time to fixation, or conversely, loss of neutral alleles is currently unresolved. Here we show that mitochondria and plastids share the same mutation rate yet plastid alleles remain in a heteroplasmic state significantly longer compared to mitochondrial alleles. By analyzing genetic variants across populations of the marine flowering plant Zostera marina and simulating organelle allele dynamics, we examine the determinants of allele segregation and allele fixation. Our results suggest that bottlenecks on the cell population, e.g., during branching or seeding, and stratification of the meristematic tissue, are important determinants of mitochondrial allele dynamics. Furthermore, we suggest that the prolonged plastid allele dynamics are due to a yet unknown active plastid partition mechanism. The dissimilarity between plastid and mitochondrial novel allele fixation at different levels of organization may manifest in differences in adaptation processes. Our study uncovers fundamental principles of organelle population genetics that are essential for further investigations of long-term evolution and molecular dating of divergence events.
PubMed: 38934796
DOI: 10.1093/molbev/msae135 -
Journal of Food and Drug Analysis Jun 2024Cell nucleus status decides the activities of corresponding cells, making its rapid and effective staining important for revealing the actual condition of biological...
Cell nucleus status decides the activities of corresponding cells, making its rapid and effective staining important for revealing the actual condition of biological environment in life science and related fields. In this study, fast staining of cell nucleus is realized by fluorescent carbon nanodots (CDs). The staining mechanism is due to the positively charged CD surface-induced cell membrane penetration, which facilitates the CD-nucleus binding via electrostatic attraction. The size of cell nucleus is easily measured with fluorescence imaging technique. In addition, the CD-based cell nucleus stain is applied for discriminating the normal and cancer cells by determining the cell-to-nucleus ratio with fluorescence images.
Topics: Carbon; Humans; Cell Nucleus; Staining and Labeling; Fluorescent Dyes; Quantum Dots; Nanoparticles; Fluorescence; Optical Imaging
PubMed: 38934693
DOI: 10.38212/2224-6614.3503 -
Archivio Italiano Di Urologia,... Jun 2024Single sperm cryopreservation (SSC) is a specific technique especially used in individuals with small numbers of sperm who suffered from non-obstructive azoospermia...
Pentoxifylline treatment as a safe method for selecting viable testicular spermatozoa before cryopreservation of a small numbers of spermatozoa in azoospermia individuals.
BACKGROUND
Single sperm cryopreservation (SSC) is a specific technique especially used in individuals with small numbers of sperm who suffered from non-obstructive azoospermia (NOA). Testicular specimens possess poor motility and low population of viable spermatozoa. Therefore, sperm selection methods such as applying pentoxifylline (PTX) may improve motility in these cases. The main aim of this study was to evaluate the protective effects of PTX on testicular spermatozoa before and after performing SSC.
METHODS
Thirty testicular samples were obtained from men with azoospermia. This study was conducted in two phases. Phase 1 evaluated the effect of PTX for sperm selection before SSC. Twenty testicular samples were divided to two experimental groups: SSC without (I) and with PTX treatment (II). For PTX treatment spermatozoa were incubated with PTX at 37°C for 30 min and only motile spermatozoa were selected for SSC. In phase 2, ten testicular samples were cryopreserved with SSC and warming procedure was carried out in droplet with and without PTX. Motility and viability rates, morphology by motile sperm organelle morphology examination (MSOME), DNA fragmentation by sperm chromatin dispersion test (SCD) and mitochondrial membrane potential (MMP) were evaluated.
RESULTS
In phase 1, post warm motility rate was higher in PTX exposed group compared to the unexposed group (25.6 ± 8.13 vs. 0.85 ± 2.1) (p > 0.00). Recovery rate, viability and morphology were not significantly different between groups. DNA integrity and MMP were also similar between both groups. In phase 2 although motility increased in PTX group compared to without PTX group (29.30 ± 12.73 vs. 1.90 ± 2.64) (p > 0.00), the viability rate was not different (70.40 ± 12.12 vs. 65.30 ± 11.87). All above mentioned parameters were similar between the two SSC groups.
CONCLUSIONS
Supplementation of testicular spermatozoa with PTX before cryopreservation increases motility and did not have adverse effects on viability, morphology, DNA integrity and MMP. PTX could be used as sperm selection method before single sperm cryopreservation, but PTX could not maintain motile the most of viable testicular sperms.
Topics: Male; Humans; Pentoxifylline; Cryopreservation; Azoospermia; Spermatozoa; Sperm Motility; Semen Preservation; DNA Fragmentation; Testis; Adult; Cell Survival; Membrane Potential, Mitochondrial
PubMed: 38934523
DOI: 10.4081/aiua.2024.12525