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The monomer, 3-hydroxybutyrate (3HB), plays an interesting role as a precursor for antibiotics, vitamins, and bioplastics such as polyhydroxybutyrates (PHB). The biotechnological production of both compounds in Escherichia coli has seen increased interest in the last decade. The central metabolite in the 3HB production pathway is acetyl-CoA, the derivative of coenzyme A (CoA). Enriching the intracellular pool of these cofactors could improve 3HB titers. In our study, we opted to increase CoA titers by upregulating pantothenate kinase (PanK), the rate limiting step in CoA biosynthetic pathway. To this end, 4 PanKs genes of different taxonomic origins (mammalian, fungal, and bacterial) were individually expressed and evaluated in 3HB producing E. coli cells. In shake flask studies, strains expressing Aspergillus nidulans PankII and Mus musculus PanK1β achieved the highest 3HB titers. In a bioreactor fermentation, the strain harboring murine PanK1β resulted in 7.6 g/L compared to 5.4 g/L of 3HB in the control strain. Although structurally different from the bacterial PankI, our study showed that eukaryotic Panks can supplement the kinase activity in prokaryotes. Expressing the exogenous PanKs offer several advantages over the host’s enzyme; PanKII is only inhibited by acetyl-CoA, for which the 3HB-production system would provide a constant metabolic sink. Additionally, PanK1β is weakly regulated by acetyl-CoA, and its activity is stimulated by free CoA. Overexpressing eukaryotic PanKs constitutes a suitable strategy for improving 3HB titers in E. coli.

Abstract Due to increasing oil prices and climate change concerns, biofuels have become increasingly important as potential alternative energy sources. However, the use of arable lands and valuable resources for the production of biofuel feedstock compromises food security and negatively affect the environment. Single cell oils (SCOs), accumulated by oleaginous yeasts, show great promise for efficient production of biofuels. However, the high production costs attributed to feedstocks or raw materials present a major limiting factor. The fermentative conversion of abundant, low-value biomass into microbial oil would alleviate this limitation. Here, we explore the feasibility of utilizing microalgae-based cell residues as feedstock for yeast oil production. We developed an efficient, single‐step enzymatic hydrolysis to generate Scenedesmus obtusiusculus hydrolysate (SH) without thermo-chemical pretreatment. With this eco-friendly process, glucose conversion efficiencies reached 90–100%. Cutaneotrichosporon oleaginosus, Cryptococcus curvatus and Rhodosporidium toruloides were cultivated on SH as sole nutrients source. Only C. oleaginosus was able to accumulate intracellular lipids, with a 35% (g lipid/g DCW) content and a yield of 3.6 g/L. Our results demonstrate the potential valorization of algal biomass into desired end-products such as biofuels. Graphic Abstract

Digitalization drives accelerated process optimization by comprehensive automation. In the advanced biofuels sector this demands automatable high-throughput processes for production strain development and downstream process performance monitoring. In that context, the unit operations of oleaginous yeast-based biodiesel production are amenable to high-throughput process development. Cutaneotrichosporon oleaginosus is a leading production strain for high-energy biofuel options, that is capable of utilizing a broad range of substrates as carbon sources, thereby generating in excess of 60% (w/w) lipids under nutrient limiting conditions. For the first time, we report on the use of fast neutron (FN) irradiation for the rapid, high-throughput genetic enhancement of an oleaginous yeast in conjunction with high-throughput selection of enhanced lipid producing C. oleaginosus mutants by cultivation in the presence of the fatty acid biosynthesis inhibitor cerulenin. Performance monitoring of improved mutants was accomplished by development of a high-throughput lipid qualification methodology based on a miniaturized, low cost Nile red based spectrofluorimetric assay. From the FN mutant library, this high-throughput strain development approach allowed identification of a C. oleaginosus variant (FN M2) displaying a 21.67% (w/v) and 22.58% (w/v) increase in biomass formation and total lipid yield compared to wild-type strain, respectively. Mutant triglyceride characterization revealed a higher content of saturated fatty acids, which is favorable with respect to biofuels production standards, determined here for the first time. This study is an initial step towards an automatable, high-throughput yeast oil optimization process that facilitates accelerated industrial deployment.

الملخص: أهداف البحث: المعروف أن الالتهابات المزمنة شائعة لدى مرضى الكلى، خاصة مرضى الفشل الكلوي في المراحل المتأخرة، حيث أنه يتزامن مع ارتفاع مؤشرات الالتهاب، التي تزيد من تدهور وظائف الكلى. والمعروف أيضا أن مرضى الفشل الكلوي قد يصاحبهم أمراض مزمنة أخرى مثل ارتفاع ضغط الدم والسكري. وبما أن العلاقة ما بين الأمراض المزمنة المصاحبة للفشل الكلوي وارتفاع مؤشرات الالتهاب لم يتم دراستها على النحو الكافي، فإن الهدف من هذه الدراسة هو تقييم مستويات مؤشرات الالتهابات في مصل الدم لدى مختلف مرضى الفشل الكلوي ودراسة العلاقة بين هذه العلامات الالتهابية والأمراض المزمنة المصاحبة له. طرق البحث: شارك في هذه الدراسة ١٤٧ مريضا، صنفوا وفقا لحالتهم المرضية: داء السكري، ومرض ارتفاع ضغط الدم، وداء السكري وارتفاع ضغط الدم معا. وتم قياس مستوى السيتوكينات، والبرتين المتفاعل سي، وعامل نخر الورم-ألفا، وإنترلوكين-١بيتا في مصل الدم لدى مختلف المجموعات من مرضى الفشل الكلوي وذلك بواسطة طريقة مقايسة المازة المناعية المرتبطة بالإنزيم. النتائج: أظهرت الدراسة أن مستويات البرتين المتفاعل سي وعامل نخر الورم-ألفا في مصل الدم مرتفعة لدى مرضى السكري، ومرضى ارتفاع ضغط الدم، وأولئك الذين كان لديهم كلا المرضين، بالمقارنة مع مرضى الفشل الكلوي غير المصحوب بأمراض مزمنة. ولم يكن هناك تغيير كبير في مستويات إنترلوكين-١بيتا بين مرضى السكري و / أو ارتفاع ضغط الدم مقارنة بالمرضى الذين لا يعانون منها. الاستنتاجات: أظهرت النتائج أن البرتين المتفاعل سي وعامل نخر الورم-ألفا يتأثران بشكل كبير بالإصابة المصاحبة لمرض الفشل الكلوي، على عكس إنترلوكين-١بيتا الذي قد يكون أكثر تأثرا بعملية غسيل الكلى نفسها حتى في غياب الأمراض المصاحبة. Abstract: Objectives: Chronic inflammatory processes are common in patients with renal disease, especially those with end-stage renal disease (ESRD), in whom inflammatory markers have been shown to increase with renal function deterioration. ESRD is usually accompanied by other chronic diseases such as hypertension and diabetes. The relationships between ESRD comorbidities and serum levels of inflammatory markers have not yet been fully understood. The aim of this study was to assess serum levels of inflammatory markers in different ESRD cohorts and to investigate the correlations between these inflammatory markers and disease comorbidities. Methods: A total of 147 patients were grouped according to their comorbid conditions: diabetic only, hypertensive only, diabetic and hypertensive, and neither diabetic nor hypertensive. Serum levels of C-reactive protein (CRP), tumour necrosis factor-alpha (TNF-α), and interleukin-1-beta (IL-1β) were investigated in different ESRD cohorts by enzyme-linked immunosorbent assay. Results: Serum CRP and TNF-α levels were high in diabetic patients (p = 0.0001), hypertensive patients (p = 0.0001), and those who had both diseases (p = 0.0001), when compared to ESRD patients without these comorbidities. There was no significant change in serum IL-1β levels between patients with diabetes mellitus and/or hypertension compared to patients who did not have these diseases. Conclusions: Our results showed that, in ESRD patients, CRP and TNF-α seem to be largely affected by patients’ comorbidities, unlike IL-1β, which might be affected more by the dialysis process even in the absence of comorbidities. الكلمات المفتاحية: السيتوكينات, داء السكري, مرض الفشل الكلوي في المراحل المتأخرة, ارتفاع ضغط الدم, Keywords: Cytokines, Diabetes, End stage kidney disease, Hypertension

Inflammation and cardiovascular disease (CVD) are common in end-stage renal disease (ESRD) patients whose vascular endothelium is in direct contact with the uremic toxins found in the blood. These toxins are believed to affect vascular injury and repair process, which is impaired in ESRD patients. The exact mechanisms behind these interactions are not clear. So, we wanted to investigate what happens at the molecular level of endothelial cells when exposed to uremic serum from ESRD patients with diabetes and/or hypertension and its effect on the expression of molecules associated with vascular injury and repair. Cultured human endothelial cells (ECV304) were incubated in the presence of normal or uremic sera from ESRD patients with diabetes and/or hypertension. The expressions of monocyte chemoattractant protein 1 (MCP-1), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) were investigated in endothelial cells (ECV304) by real-time PCR and ELISA. The expression of MCP-1, VEGF, and SDF-1 was elevated in endothelial cells upon exposure to uremic sera from ESRD patients with diabetes and/or hypertension when compared with cells treated with healthy serum. MCP-1 expression in endothelial cells treated with uremic serum from ESRD patients with hypertension only was significantly increased compared with its expression in other cohorts. Exposure of endothelial cells to uremic serum causes endothelial injury and inflammation characterized by an increase in MCP-1 expression. This injury activates the initiation of vascular repair process in these cells by increasing the expression of VEGF and SDF-1. These molecules can be important biomarkers of chronic kidney disease-associated CVD.

Inflammation and cardiovascular disease (CVD) are common in end-stage renal disease (ESRD) patients whose vascular endothelium is in direct contact with the uremic toxins found in the blood. These toxins are believed to affect vascular injury and repair process, which is impaired in ESRD patients. The exact mechanisms behind these interactions are not clear. So, we wanted to investigate what happens at the molecular level of endothelial cells when exposed to uremic serum from ESRD patients with diabetes and/or hypertension and its effect on the expression of molecules associated with vascular injury and repair. Cultured human endothelial cells (ECV304) were incubated in the presence of normal or uremic sera from ESRD patients with diabetes and/or hypertension. The expressions of monocyte chemoattractant protein 1 (MCP-1), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) were investigated in endothelial cells (ECV304) by real-time PCR and ELISA. The expression of MCP-1, VEGF, and SDF-1 was elevated in endothelial cells upon exposure to uremic sera from ESRD patients with diabetes and/or hypertension when compared with cells treated with healthy serum. MCP-1 expression in endothelial cells treated with uremic serum from ESRD patients with hypertension only was significantly increased compared with its expression in other cohorts. Exposure of endothelial cells to uremic serum causes endothelial injury and inflammation characterized by an increase in MCP-1 expression. This injury activates the initiation of vascular repair process in these cells by increasing the expression of VEGF and SDF-1. These molecules can be important biomarkers of chronic kidney disease-associated CVD.

In the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-02254-7) contains supplementary material, which is available to authorized users.

Aims The main function of the colon is water and electrolyte absorption. Total colectomy eliminates this colonic function and may alter the absorptive capacity of the small intestine for nutrients. This study examines the effect of total colectomy on jejunal glucose absorption and investigates the potential role of aldosterone in mediating the alterations in glucose uptake post-colectomy using the aldosterone antagonist spironolactone. Main methods Total colectomy with ileo-rectal anastomosis was performed on anesthetized rats. Sham rats were identically handled without colon resection. Two days post-surgery, groups of colectomized rats were injected with either a daily subcutaneous dose of spironolactone or sesame oil for 12 days. Body weight changes and food and water intake were measured in all experimental groups. Glucose absorption was measured by in-vivo single pass perfusion in the rat jejunum of control, sham, colectomized, colectomized with spironolactone, and colectomized with sesame oil treatment. Na/K ATPase, SGK1, SGLT1 and GLUT2 expressions were determined in jejunal mucosa in control, colectomized and colectomized/spironolactone injected rats by Western blot analysis. Histological assessment was performed on jejunal sections in control and colectomized groups. Key findings Glucose absorption significantly increased in colectomized rats with an observed increase in Na/K ATPase and SGK1 expression. No significant expression change in SGLT1 and GLUT2 was detected in the jejunum in colectomized rats. Spironolactone, however, significantly decreased the glucose uptake post-colectomy and normalized Na/K ATPase and SGK1 expression. Significance Our results suggest that jejunal glucose uptake increases post-colectomy as a possible consequence of an aldosterone-mediated function.