Transcriptome and Metabolome Profiling of a Novel Isolate Chlorella sorokiniana G32 (Chlorophyta) Displaying Enhanced Starch Accumulation at High Growth Rate Under Mixotrophic Condition

Chlorella sorokiniana is one of the most productive microalgal species with a high potential for the production of biofuels and other high value-added molecules. Many studies have focused on its capability of mixotrophic growth using reduced organic carbon and growth pattern shift between autotrophic and mixotrophic conditions. In this study, we investigated growth patterns of a novel isolate, C. sorokiniana G32, under mixotrophic growth conditions supplemented with a low level (1.25 g L–1) and a high level (5 g L–1) of glucose. Physiological, transcriptomic (i.e., RNA-seq), and metabolomic (i.e., LC-MS/MS) methods were used. We showed that peak growth based on OD680nm absorbance is ∼4-fold higher with high glucose vs. low glucose supplementation. Photosynthetic efficiency (Fv/Fm) in G32 mixotrophic cultures with high or low glucose supplementation remains identical to that of G32 phototrophic growth. We also found that the conversion rate between absorbance-based cell density and cell dry weight with high glucose supplementation was lower than with low glucose. This suggests that more cell biomass is produced under high glucose treatment than with low glucose. The result was confirmed via sucrose density gradient centrifugation. It is likely that accumulation of high concentration of starch may account for this effect. Transcriptomic analysis of G32 cultures (i.e., via RNA-seq) in response to reciprocal change of glucose levels reveals that expression of a subset of differentially expressed genes (DEGs) is correlated with the amount of glucose supplementation. These DEGs are designated as glucose-specific responsive (GSR) genes. GSR genes are enriched for a number of energy metabolic pathways. Together with metabolomics data (i.e., LC-MS/MS), we show that under high-level supplementation, glucose is preferentially oxidized through an oxidative pentose phosphate pathway. Collectively, our results indicate the mechanism of regulation of glucose assimilation and energy metabolism in G32 under mixotrophic conditions with different levels of glucose supplementation revealed by transcriptomic and metabolomic analyses. We propose that C. sorokiniana G32 has the potential for the production of high value-added molecules.

Adenosine A2A Receptor in Bone Marrow-Derived Cells Mediated Macrophages M2 Polarization via PPARγ-P65 Pathway in Chronic Hypoperfusion Situation

Background: The role of adenosine A2A receptor (A2AR) in the ischemic white matter damage induced by chronic cerebral hypoperfusion remains obscure. Here we investigated the role of A2AR in the process of macrophage polarizations in the white matter damage induced by chronic cerebral hypoperfusion and explored the involved signaling pathways.Methods: We combined mouse model and macrophage cell line for our study. White matter lesions were induced in A2AR knockout mice, wild-type mice, and chimeric mice generated by bone marrow cells transplantation through bilateral common carotid artery stenosis. Microglial/macrophage polarization in the corpus callosum was detected by immunofluorescence. For the cell line experiments, RAW264.7 macrophages were treated with the A2AR agonist CHS21680 or A2AR antagonist SCH58261 for 30 min and cultured under low-glucose and hypoxic conditions. Macrophage polarization was examined by immunofluorescence. The expression of peroxisome proliferator activated receptor gamma (PPARγ) and transcription factor P65 was examined by western blotting and real-time polymerase chain reaction (RT-PCR). Inflammatory cytokine factors were assessed by enzyme-linked immunosorbent assay (ELISA) and RT-PCR.Results: Both global A2AR knockout and inactivation of A2AR in bone marrow-derived cells enhanced M1 marker expression in chronic ischemic white matter lesions. Under low-glucose and hypoxic conditions, CGS21680 treatment promoted macrophage M2 polarization, increased the expression of PPARγ, P65, and interleukin-10 (IL-10) and suppressed the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The CGS21680-induced upregulation of P65 and IL-10 was abolished in macrophages upon PPARγ knockdown. The downregulation of TNF-α and IL-1β by CGS21680 was less affected by PPARγ knockdown.Conclusions: In the cerebral hypoperfusion induced white matter damage, A2AR signaling in bone marrow-derived cells induces macrophage M2 polarization and increases the expression of the anti-inflammatory factor IL-10 via the PPARγ-P65 pathway, both of which might explain its neuroprotective effect.

A Low-Glucose Eating Pattern Improves Biomarkers of Postmenopausal Breast Cancer Risk: An Exploratory Secondary Analysis of a Randomized Feasibility Trial

Postmenopausal breast cancer is the most common obesity-related cancer death among women in the U.S. Insulin resistance, which worsens in the setting of obesity, is associated with higher breast cancer incidence and mortality. Maladaptive eating patterns driving insulin resistance represent a key modifiable risk factor for breast cancer. Emerging evidence suggests that time-restricted feeding paradigms (TRF) improve cancer-related metabolic risk factors; however, more flexible approaches could be more feasible and effective. In this exploratory, secondary analysis, we identified participants following a low-glucose eating pattern (LGEP), defined as consuming energy when glucose levels are at or below average fasting levels, as an alternative to TRF. Results show that following an LGEP regimen for at least 40% of reported eating events improves insulin resistance (HOMA-IR) and other cancer-related serum biomarkers. The magnitude of serum biomarkers changes observed here has previously been shown to favorably modulate benign breast tissue in women with overweight and obesity who are at risk for postmenopausal breast cancer. By comparison, the observed effects of LGEP were similar to results from previously published TRF studies in similar populations. These preliminary findings support further testing of LGEP as an alternative to TRF and a postmenopausal breast cancer prevention strategy. However, results should be interpreted with caution, given the exploratory nature of analyses.

Determination of the Role of the Distal Outflow Pathway Tissue in Glucocorticoid-induced Ocular Hypertension

Introduction Prolonged application of glucocorticoids (GCs) induces ocular hypertension (OHT) and glaucoma. This increased intraocular pressure (IOP) is due to pathological changes in the trabecular meshwork (TM) outflow pathway tissues including impaired cell functions and extracellular matrix deposition. The changes and role of the TM in GC-induced OHT have been well studied. However, the role of the tissues distal to the TM (distal outflow tissues) is unclear. This study aims to further uncover the role of distal outflow tissue in GC-induced OHT using a novel perfusion organ culture (POC) model. Methods Huma corneal rims tissues were attached to 3D printed transparent perfusion plates using a combination of thin and thick glues. The artificial anterior chamber was perfused with DMEM-low glucose medium at 2ul/min to mimic aqueous humor production, and IOP was recorded using pressure transducers and a computerized system. To determine the role of distal tissue in GC-induced IOP changes, the TM tissue was carefully removed from both eyes, and one eye was treated with ethanol (EtOH) and the fellow eye with dexamethasone (DEX). Results The model was validated through a comparison of the IOP and TM stiffness of glue contaminated to non-contaminated corneal rims. The glue contaminated rim showed highly increased IOP and TM stiffness while the non-contaminated rim showed normal values. After validation, the TM was removed from paired corneal rims. One rim was treated with 100nM DEX and the fellow rim with 0.1% EtOH. The DEX treated rim showed increase in IOP while the EtOH control showed little change. Conclusion We created a novel corneal rim perfusion culture model for the study of GC-induced OHT. This model showed promising results of distal outflow involvement in glucocorticoid induced ocular hypertension. Further studies are needed to elucidate the role of distal outflow tissues in GC responsiveness in the eye.

Lactic Acid Transport Mediated by Aquaporin-9: Implications on the Pathophysiology of Preeclampsia

Aquaporin-9 (AQP9) expression is significantly increased in preeclamptic placentas. Since feto-maternal water transfer is not altered in preeclampsia, the main role of AQP9 in human placenta is unclear. Given that AQP9 is also a metabolite channel, we aimed to evaluate the participation of AQP9 in lactate transfer across the human placenta. Explants from normal term placentas were cultured in low glucose medium with or without L-lactic acid and in the presence and absence of AQP9 blockers (0.3 mM HgCl2 or 0.5 mM Phloretin). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase release. Apoptotic indexes were analyzed by Bax/Bcl-2 ratio and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling assay. Heavy/large and light/small mitochondrial subpopulations were obtained by differential centrifugation, and AQP9 expression was detected by Western blot. We found that apoptosis was induced when placental explants were cultured in low glucose medium while the addition of L-lactic acid prevented cell death. In this condition, AQP9 blocking increased the apoptotic indexes. We also confirmed the presence of two mitochondrial subpopulations which exhibit different morphologic and metabolic states. Western blot revealed AQP9 expression only in the heavy/large mitochondrial subpopulation. This is the first report that shows that AQP9 is expressed in the heavy/large mitochondrial subpopulation of trophoblasts. Thus, AQP9 may mediate not only the lactic acid entrance into the cytosol but also into the mitochondria. Consequently, its lack of functionality in preeclamptic placentas may impair lactic acid utilization by the placenta, adversely affecting the survival of the trophoblast cells and enhancing the systemic endothelial dysfunction.

An emerging simple and effective approach to increase the productivity of thraustochytrids microbial lipids by regulating glycolysis process and triacylglycerols’ decomposition

Background The oleaginous microorganism Schizochytrium sp. is widely used in scientific research and commercial lipid production processes. However, low glucose-to-lipid conversion rate (GLCR) and low lipid productivity of Schizochytrium sp. restrict the feasibility of its use. Results Orlistat is a lipase inhibitor, which avoids triacylglycerols (TAGs) from hydrolysis by lipase. TAGs are the main storage forms of fatty acids in Schizochytrium sp. In this study, the usage of orlistat increased the GLCR by 21.88% in the middle stage of fermentation. Whereas the productivity of lipid increased 1.34 times reaching 0.73 g/L/h, the saturated fatty acid and polyunsaturated fatty acid yield increased from 21.2 and 39.1 to 34.9 and 48.5 g/L, respectively, indicating the advantages of using a lipase inhibitor in microbial lipids fermentation. Similarly, the system was also successful in Thraustochytrid Aurantiochytrium. The metabolic regulatory mechanisms stimulated by orlistat in Schizochytrium sp. were further investigated using transcriptomics and metabolomics. The results showed that orlistat redistributed carbon allocation and enhanced the energy supply when inhibiting the TAGs’ degradation pathway. Therefore, lipase in Schizochytrium sp. prefers to hydrolyze saturated fatty acid TAGs into the β-oxidation pathway. Conclusions This study provides a simple and effective approach to improve lipid production, and makes us understand the mechanism of lipid accumulation and decomposition in Schizochytrium sp., offering new guidance for the exploitation of oleaginous microorganisms.