Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter.

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (P AOX1 ) and/or the constitutive GAP promoter (P GAP ). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (PDH) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical P GAP was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under PDH outperformed P GAP controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that PDH is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for PDH under different feeding and osmolarity conditions. In all of them the PDH controlled process outperformed the P GAP one in regard to CalB titer. The best PDH approach reached 3.6-fold more specific productivity than P GAP fed-batch at low µ. Compared to the optimum approach for P GAP -based process, the best PDH fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, PDH is an inducible promoter that exhibited a non-coupled growth regulation showing high performance, which provides a methanol-free additional solution to the usual growth-coupled systems for RPP. Thus, this novel system emerges as a potential alternative for K. phaffii RPP bioprocess and for revaluing crude glycerol, promoting the transition towards a circular economy.

Psychological distress in patients undergoing surgery for urological cancer: A single centre cross-sectional study.

PURPOSE: Interest in the disease-specific psychological well-being of patients with cancer has increased, and it has been estimated that less than half of all patients with cancer are properly identified and subsequently treated for anxiety or depression. The aim of this study is to evaluate psychological distress in uro-oncological patients undergoing different surgeries: radical cystectomy, radical prostatectomy, radical nephrectomy, or transurethral resection (TUR) before the surgery. MATERIALS AND METHODS: We performed a cross-sectional study in consecutively enrolled patients with bladder, kidney, or prostate cancer, scheduled for surgery. Demographic data, socioeconomic status, education level, and diagnoses were recorded. Patients with a previous diagnosis of depression or anxiety were excluded. We evaluated the level of clinically meaningful depression and anxiety assessed by 2 tools: the Hospital Anxiety and Depression Scale (HADS; score ≥8 presence of anxiety and depression; score ≥11 clinical anxiety and depression) and the State-Trait Anxiety Inventory (STAI). To determine variables related to depression and anxiety among the demographic variables, logistic regression analyses were conducted, with P<0.05 considered as statistically significant. RESULTS: A total of 207 patients were recruited, completed the questionnaires and were included in the study. Patients presented a mean age of 70.8 (±10.8) years, 89% were males (n = 184) and 19% of patients presented previous cancer. The majority of patients underwent surgery for bladder tumors (60.4%) and the most common type of surgery was TUR. The most frequent procedures were performed for bladder tumors (60.4%), being TUR the most common type of surgery (52.7%) followed by radical prostatectomy (24.6%). Mean STAI-State score was 19.3 (±10.3), and mean STAI-Trait score was 18.4 (±11.9) points. Clinical levels of anxiety and depression (HADS ≥ 11 points) were found in 19 (9.8%) and 7 (3.6%) cases. And HADS anxiety 8 to 10 points was present in 14.5% (n = 28) and HADS depression 8 to 10 points in 5.7% (n = 11) of the sample, representing presence of psychological distress. Female patients showed a higher level of anxiety and STAI-Trait compared to males. CONCLUSION: The present results show that our patients had lower levels of anxiety and depression than those described in the literature. Sex, tumor type, and surgical approach were significantly related to psychological distress in patients undergoing surgery for urological cancer. Females and patients with kidney tumor and patients undergoing radical nephrectomy presented higher levels of anxiety. Patients with radical cystectomy showed a higher level of STAI-State compared with other surgeries.

Modulation of SHBG binding to testosterone and estradiol by sex and morbid obesity.

OBJECTIVE: Sex hormone-binding globulin (SHBG) binds and transports testosterone and estradiol in plasma. The possibility that SHBG is a mixture of transporting proteins has been postulated. We analyzed in parallel the effects of obesity status on the levels and binding capacity of circulating SHBG and their relationship with testosterone and estradiol. DESIGN: Anthropometric measures and plasma were obtained from apparently healthy young (i.e. 35 ± 7 years) premenopausal women (n = 32) and men (n = 30), with normal weight and obesity (BMI >30 kg/m2). METHODS: SHBG protein (Western blot), as well as the plasma levels of testosterone, estradiol, cortisol and insulin (ELISA) were measured. Specific binding of estradiol and testosterone to plasma SHBG was analyzed using tritium-labeled hormones. RESULTS: Significant differences in SHBG were observed within the obesity status and gender, with discordant patterns of change in testosterone and estradiol. In men, testosterone occupied most of the binding sites. Estrogen binding was much lower in all subjects. Lower SHBG of morbidly obese (BMI >40 kg/m2) subjects affected testosterone but not estradiol. The ratio of binding sites to SHBG protein levels was constant for testosterone, but not for estradiol. The influence of gender was maximal in morbid obesity, with men showing the highest binding/SHBG ratios. CONCLUSIONS: The results reported here are compatible with SHBG being a mixture of at least two functionally different hormone-binding globulins, being affected by obesity and gender and showing different structure, affinities for testosterone and estradiol and also different immunoreactivity.