Growing a circular economy with fungal biotechnology: a white paper.

Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation's sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.

Synthetic ACTH in High Risk Patients with Idiopathic Membranous Nephropathy: A Prospective, Open Label Cohort Study.

New therapeutic agents are warranted in idiopathic membranous nephropathy. Synthetic ACTH may be advantageous with reported remission rates up to 85% and few side effects. We conducted a prospective open label cohort study from 2008 till 2010 (NCT00694863). We prospectively selected patients with idiopathic membranous nephropathy and high risk for progression (defined as ßeta-2-microglobulin (ß2m) excretion of >500 ng/min). For comparison, we selected matched historical controls treated with cyclophosphamide. The prospectively selected patients received intramuscular injections of synthetic ACTH during 9 months (maximal dose 1 mg twice a week). The primary endpoints concerned the feasibility and incidence of remissions as a primary event. Secondary endpoints included side effects of treatment and the incidence of remissions and relapses at long-term follow-up. Twenty patients (15 men) were included (age 54±14 years, serum creatinine 104 µmol/l [IQR 90­113], urine protein:creatinine ratio 8.7 g/10 mmol creatinine [IQR 4.3­11.1]). Seventeen patients (85%) completed treatment. 97% of injections were administered correctly. Cumulative remission rate was 55% (complete remission in 4 patients, partial remission 7 patients). In a group of historical controls treated with cyclophosphamide and steroids, 19 of 20 patients (95%) developed a remission (complete remission in 13 patients, partial remission in 6 patients) (p<0.01). The main limitation of our study is its small size and the use of a historical control group. We show that treatment with intramuscular injections of synthetic ACTH is feasible. Our data suggest that synthetic ACTH is less effective than cyclophosphamide in inducing a remission in high risk patients with idiopathic membranous nephropathy. The use of synthetic ACTH was also associated with many adverse events. Therefore, we advise against synthetic ACTH as standard treatment in membranous nephropathy.

Survival, comorbidities and joint damage 11 years after the COBRA combination therapy trial in early rheumatoid arthritis.

BACKGROUND: COBRA (for 'COmbinatie therapie Bij Rheumatoide Artritis') combination therapy is effective for the treatment of rheumatoid arthritis (RA), but long-term safety is unknown. This study evaluates survival, comorbidities and joint damage in the original COBRA trial cohort. METHODS: In the COBRA trial, 155 patients with early RA were treated with sulfasalazine (SSZ) monotherapy (SSZ group) or a combination of step-down prednisolone, methotrexate (MTX) and SSZ (COBRA group). The current 11-year follow-up study of the COBRA trial invited all original patients and performed protocollised scrutiny of clinical records, questionnaires, physical examination, laboratory and imaging tests. RESULTS: In all, 152 out of 155 patients yielded at least partial data. After a mean of 11 years follow-up, 18 (12%) patients had died, 6 COBRA patients and 12 SSZ patients, HR 0.57 (95% CI 0.21 to 1.52). Treatment for hypertension was significantly more prevalent in the COBRA group (p=0.02) with similar trends for diabetes and cataract. Conversely, hypercholesterolaemia, cancer and infection showed a trend in favour of COBRA. Other comorbidities such as cardiovascular disease and fractures appeared in similar frequency. Radiographic findings suggest as a minimum sustained benefit for COBRA therapy, that is, difference in joint damage but similar subsequent progression rates after 5 years. Imputation to compensate for selective dropout suggests increasing benefit for COBRA, that is, difference in yearly progression rates similar to that seen in the first 5 years of follow-up. CONCLUSIONS: After 11 years, initial COBRA combination therapy resulted in numerically lower mortality and similar prevalence of comorbidity compared with initial SSZ monotherapy. In addition, lower progression of joint damage suggests long-term disease modification.

Impact of vitamin D dose on biochemical parameters in patients with secondary hyperparathyroidism receiving cinacalcet.

BACKGROUND/AIMS: The calcimimetic cinacalcet (Mimpara/Sensipar) simultaneously lowers parathyroid hormone (PTH), phosphorus (P) and calcium (Ca) levels in patients with secondary hyperparathyroidism. The OPTIMA study demonstrated that cinacalcet and adjusted doses of vitamin D maximized control of these parameters. This post-hoc analysis of OPTIMA data assessed the impact of reducing or increasing the dose of concomitant vitamin D on PTH, P and Ca in patients receiving cinacalcet. METHODS: Dialysis patients with mean baseline intact PTH (iPTH) 300-800 pg/ml (31.8-84.8 pM) received doses of cinacalcet titrated to achieve an iPTH of 150-300 pg/ml (15.9-31.8 pM). The dose of vitamin D could then be decreased to further reduce serum P or Ca, or increased/initiated to further decrease PTH levels if iPTH >300 pg/ml or to increase Ca if Ca <8.0 mg/dl (2.0 mM). RESULTS: Vitamin D dose was assessed for 345 patients during a 23-week period. A total of 91 and 129 patients had an increase or decrease in vitamin D dose, respectively. By study end, mean iPTH, P, and Ca were similar in both vitamin D groups, although there were differences in biochemical parameters between groups at the start of the study. There were statistically significant reductions from baseline to study end in iPTH and Ca in both groups (p < 0.001). Although P was significantly reduced by week 23 in the group in which vitamin D dose was decreased (p = 0.007), the reduction in P was less and did not achieve significance in the group in which vitamin D dose was increased (p = 0.71). CONCLUSIONS: After initiating cinacalcet, the dose of vitamin D can be adjusted to maximize reductions in PTH, P and Ca; however, vitamin D-induced decreases in PTH need to be balanced with the diminished response in P and Ca.

The OPTIMA study: assessing a new cinacalcet (Sensipar/Mimpara) treatment algorithm for secondary hyperparathyroidism.

BACKGROUND AND OBJECTIVES: Cinacalcet, a novel calcimimetic, targets the calcium-sensing receptor to lower parathyroid hormone (PTH), calcium, and phosphorus levels in dialysis patients with secondary hyperparathyroidism (SHPT). This study compared the efficacy of a cinacalcet-based regimen with unrestricted conventional care (vitamin D and phosphate binders) for achieving the stringent National Kidney Foundation Kidney Disease Outcomes Quality Initiative (KDOQI) targets for dialysis patients. STUDY DESIGN: In this multicenter, open-label study, hemodialysis patients with poorly controlled SHPT were randomized to receive conventional care (n = 184) or a cinacalcet-based regimen (n = 368). Doses of cinacalcet, vitamin D sterols, and phosphate binders were adjusted during a 16-wk dose-optimization phase with the use of algorithms that allowed cinacalcet to be used with adjusted doses of vitamin D. The primary end point was the proportion of patients with mean intact PTH < or =300 pg/ml during a 7-wk efficacy assessment phase. RESULTS: A higher proportion of patients receiving the cinacalcet-based regimen versus conventional care achieved the targets for PTH (71% versus 22%, respectively; P < 0.001), Ca x P (77% versus 58%, respectively; P < 0.001), calcium (76% versus 33%, respectively; P < 0.001), phosphorus (63% versus 50%, respectively; P = 0.002), and PTH and Ca x P (59% versus 16%, respectively, P < 0.001), and allowed a 22% reduction in vitamin D dosage in patients receiving vitamin D at baseline. Achievement of targets was greatest in patients with less severe disease (intact PTH range, 300 to 500 pg/ml) and the cinacalcet dose required was lower in these patients (median = 30 mg/d). CONCLUSIONS: Compared with conventional therapy, a cinacalcet-based treatment algorithm increased achievement of KDOQI treatment targets in dialysis patients in whom conventional therapy was no longer effective in controlling this disease.

Increased production of chymosin by glycosylation.

Filamentous fungi are well known in the industry as producers of large amounts of extracellular proteins. However, production levels of heterologous proteins are often disappointing low. In this paper it is shown that increasing glycosylation is a powerful strategy for increasing production levels of chymosin in filamentous fungi. Two different concepts based on glycosylation were tested. First, we improved a poorly used N-glycosylation site within the prochymosin molecule. The resulting highly glycosylated chymosin molecule was expressed in Aspergillus niger. It was shown that production of the glycosylated protein was much more efficient, giving a yield increase of more than 100% compared to production of the native chymosin molecule. In an alternative strategy the N-glycosylation site was located outside of the native chymosin molecule, on a linker separating prochymosin from its carrier molecule. Also in this case significantly increased production levels were obtained. This strategy might offer a powerful tool for increasing production levels of other heterologous proteins as well.

Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk.

Enzymatic milk coagulation for cheese manufacturing involves the cleavage of the scissile bond in kappa-casein by an aspartic acid protease. Bovine chymosin is the preferred enzyme, combining a strong clotting activity with a low general proteolytic activity. In the present study, we report expression and enzymatic properties of recombinant camel chymosin expressed in Aspergillus niger. Camel chymosin was shown to have different characteristics than bovine chymosin. Camel chymosin exhibits a 70% higher clotting activity for bovine milk and has only 20% of the unspecific protease activity for bovine chymosin. This results in a sevenfold higher ratio of clotting to general proteolytic activity. The enzyme is more thermostable than bovine chymosin. Kinetic analysis showed that half-saturation is achieved with less than 50% of the substrate required for bovine chymosin and turnover rates are lower. While raw camel milk cannot be clotted with bovine chymosin, a high clotting activity was found with camel chymosin.

The effects of control of thermal balance on vascular stability in hemodialysis patients: results of the European randomized clinical trial.

BACKGROUND: Many reports note that the use of cool dialysate has a protective effect on blood pressure during hemodialysis (HD) treatments. However, formal clinical trials in which dialysate temperature is tailored to the body temperature of appropriately selected hypotension-prone patients are lacking. METHODS: We investigated the effect of thermal control of dialysate on hemodynamic stability in hypotension-prone patients selected from 27 centers in nine European countries. Patients were eligible for the study if they had symptomatic hypotensive episodes in 25% or more of their HD sessions, assessed during a prospective screening phase over 1 month. The study is designed as a randomized crossover trial with two phases and two treatment arms, each phase lasting 4 weeks. We used a device allowing the regulation of thermal balance (Blood Temperature Monitor; Fresenius Medical Care, Bad Homberg, Germany), by which we compared a procedure aimed at preventing any transfer of thermal energy between dialysate and extracorporeal blood (thermoneutral dialysis) with a procedure aimed at keeping body temperature unchanged (isothermic dialysis). RESULTS: One hundred sixteen HD patients were enrolled, and 95 patients completed the study. During thermoneutral dialysis (energy flow rate: DeltaE = -0.22 +/- 0.29 kJ/kg x h), 6 of 12 treatments (median) were complicated by hypotension, whereas during isothermic dialysis (energy flow rate: DeltaE = -0.90 +/- 0.35 kJ/kg x h), the median decreased to 3 of 12 treatments (P < 0.001). Systolic and diastolic blood pressures and heart rate were more stable during the latter procedure. Isothermic dialysis was well tolerated by patients. CONCLUSION: Results show that active control of body temperature can significantly improve intradialytic tolerance in hypotension-prone patients.