Building Yarrowia lipolytica Cell Factories for Advanced Biomanufacturing: Challenges and Solutions.

Microbial cell factories have shown great potential for industrial production with the benefit of being environmentally friendly and sustainable. Yarrowia lipolytica is a promising and superior non-model host for biomanufacturing due to its cumulated advantages compared to model microorganisms, such as high fluxes of metabolic precursors (acetyl-CoA and malonyl-CoA) and its naturally hydrophobic microenvironment. However, although diverse compounds have been synthesized in Y. lipolytica cell factories, most of the relevant studies have not reached the level of industrialization and commercialization due to a number of remaining challenges, including unbalanced metabolic flux, conflict between cell growth and product synthesis, and cytotoxic effects. Here, various metabolic engineering strategies for solving the challenges are summarized, which is developing fast and extremely conducive to rational design and reconstruction of robust Y. lipolytica cell factories for advanced biomanufacturing. Finally, future engineering efforts for enhancing the production efficiency of this platform strain are highlighted.

Analysis of growth dynamics in five different media and metabolic phenotypic characteristics of Piriformospora indica.

Piriformospora indica is an important endophytic fungus with broad potential for alleviating biotic and abiotic stress on host plants. This study monitored the growth dynamics of P. indica on five commonly used artificial media for microorganisms and analyzed its metabolic characteristics using Biolog Phenotype Microarray (PM) technology. The results showed that P. indica grew fastest on Potato Dextrose Agar (PDA), followed by Kidney Bean Agar (KBA), Alkyl Ester Agar (AEA), Oatmeal Agar (OA), and Luria-Bertani Agar (LB), and the most suitable medium for spore production was OA. Using Biolog PM1-10, 950 metabolic phenotypes of P. indica were obtained. P. indica could metabolize 87.89% of the tested carbon sources, 87.63% of the tested nitrogen sources, 96.61% of the tested phosphorus sources, and 100% of the tested sulfur sources. P. indica displayed 92 kinds of tested biosynthetic pathways, and it could grow under 92 kinds of tested osmotic pressures and 88 kinds of tested pH conditions. PM plates 1-2 revealed 43 efficient carbon sources, including M-Hydroxyphenyl acid, N-Acetyl-D-Glucosamine, Tyramine, Maltotrios, α-D-Glucosine, I-Erythritol, L-Valine, D-Melezitose, D-Tagatose, and Turanose. PM plates 3,6-8 indicated 170 efficient nitrogen sources, including Adenosine, Inosine Allantoin, D, L-Lactamide, Arg-Met, lle-Trp, Ala-Arg, Thr-Arg, Trp-Tyr, Val-Asn, Gly-Gly-D-Leu, Gly-Gly-Phe, and Leu-Leu-Leu. This study demonstrates that P. indica can metabolize a variety of substrates, such as carbon and nitrogen sources, and has a wide range of environmental adaptability. The growth dynamics on artificial culture media and metabolic phenotypes of P. indica can be used to investigate its biological characteristics, screen for more suitable growth and sporulation conditions, and elucidate the physiological mechanisms that enhance the stress resistance of host plants. This study provides a theoretical basis for its better application in agriculture.

[Differences of blood plasma renin activity, angiotensin II and aldosterone levels in essential or secondary hypertension].

OBJECTIVE: To study on the difference of plasma renin activity (PRA), angiotensin II (Ang II), and aldosterone levels in patients with essential hypertension (EH) or primary aldosteronism (PA) or pheochromocytoma (PHEO), and to analyze the sensitivity and specificity on the diagnosis of PA among patients with hypertension with aldosterone/PRA ratio (ARR). METHODS: The plasma aldosterone, Ang II and PRA concentrations in supine and upright positions were measured by radioimmunoassay from 413 patients including idiopathic hyperaldosteronism (IHA, n = 111), aldosterone-producing adenoma (APA, n = 118), PHEO (n = 98) and EH (n = 86). ARR was calculated. RESULTS: Plasma aldosterone concentrations in both of supine and upright positions in PHEO group [374 (294, 465) pmol/L and 629 (449, 997) pmol/L] and PA group [471 (346, 632) pmol/L and 673 (499, 825) pmol/L] were higher than those in EH group [277 (224, 332) pmol/L and 427 (341, 501) pmol/L] (P < 0.01). They were also higher in APA group [576 (416, 731) pmol/L and 726 (554, 906) pmol/L] than those in IHA group [399 (313, 504) pmol/L and 609 (485, 776) pmol/L] (P < 0.01). Ang II levels in both positions were lower in PA group [43.2 (26.4, 74.4) ng/L and 60.1 (38.5, 103.6) ng/L] than in EH group [56.7 (43.3, 78.9) ng/L and 84.3 (61.3, 108.4) ng/L] or PHEO group [54.3 (29.9, 101.5) ng/L and 102.8 (49.9, 167.0) ng/L] (all P values < 0.01), and there was no difference between IHA and APA group (P > 0.05). The PRA level in both positions of each group were PHEO group [0.3 (0.2, 1.0) µg · L(-1) · h(-1) and 1.4 (0.6, 3.4) µg · L(-1) · h(-1)] > EH group [0.2 (0.1, 0.4) µg · L(-1) · h(-1) and 0.6 (0.4, 1.0) µg · L(-1) · h(-1)] (P < 0.01) > PA group [0.1 (0.1, 0.1) µg · L(-1) · h(-1) and 0.2 (0.1, 0.3) µg · L(-1) · h(-1)] (P < 0.01), and APA group [0.1 (0.1, 0.1) µg · L(-1) · h(-1) and 0.1 (0.1, 0.3) µg · L(-1) · h(-1)] < IHA group [0.1 (0.1, 0.2) µg · L(-1) · h(-1) and 0.2 (0.1, 0.3) µg · L(-1) · h(-1)] (supine P < 0.01; upright P < 0.05). APA was divided into 2 types with renin-Ang II-responsive APA (n = 26) and unresponsive APA (n = 92). The plasma aldosterone concentration was lower in supine position but higher in upright position in renin-Ang II-responsive APA than in unresponsive APA patients. ARR in upright was higher in PA group (P < 0.01) but lower in PHEO group (P < 0.05) compared with EH. ARR was higher in APA than in IHA (P < 0.01). The sensitivity and specificity of ARR as 40 (aldosterone unit: ng/dl; PRA unit: µg · L(-1) · h(-1); its value should multiply 27.7 when transferred to pmol/L, simili) were 93% and 76%, respectively. CONCLUSION: The levels of PRA, Ang II and aldosterone from patients with EH, PA and PHEO are significant different. ARR as 40 in upright position could be used for PA screening cutoff point.

Advancing Yarrowia lipolytica as a superior biomanufacturing platform by tuning gene expression using promoter engineering.

Yarrowia lipolytica is recognized as an excellent non-conventional yeast in the field of biomanufacturing, where it is used as a host to produce oleochemicals, terpenes, organic acids, polyols and recombinant proteins. Consequently, metabolic engineering of this yeast is becoming increasingly popular to advance it as a superior biomanufacturing platform, of which promoters are the most basic elements for tuning gene expression. Endogenous promoters of Yarrowia lipolytica were reviewed, which are the basis for promoter engineering. The engineering strategies, such as hybrid promoter engineering, intron enhancement promoter engineering, and transcription factor-based inducible promoter engineering are described. Additionally, the applications of Yarrowia lipolytica promoter engineering to rationally reconstruct biosynthetic gene clusters and improve the genome-editing efficiency of the CRISPR-Cas systems were reviewed. Finally, research needs and future directions for promoter engineering are also discussed in this review.

Magnetic Resonance Imaging (MRI) Phenotypes May Provide Additional Information for Risk Stratification for Encapsulated Papillary Carcinoma of the Breast.

BACKGROUND: Encapsulated papillary carcinoma (EPC) of the breast is a rare entity. EPC can be underappreciated on percutaneous biopsy, which may require additional procedures if invasion is not recognized preoperatively. We aimed to investigate the magnetic resonance imaging (MRI) phenotypes correlated with preoperative pathological risk stratification for clinical guidance. MATERIALS AND METHODS: The preoperative MRI scans of 30 patients diagnosed with 36 EPCs in multiple centers between August 2015 and February 2020 were reviewed by two breast radiologists. According to the WHO classification published in 2019, EPCs were classified into two pathological subtypes: encapsulated papillary carcinoma and encapsulated papillary carcinoma with invasion. Clinicopathological analysis of the two subtypes and MR feature analysis were performed. RESULTS: Evaluation of the MRI phenotypes and pathological subtype information revealed that not circumscribed (P=0.04) was more common in EPCs with invasion than in EPCs. There was a significant difference in the age of patients (P=0.05), and the risk increased with age. The maximum diameter of the tumor increased with tumor risk, but there was no significant difference (P=0.36). Nearly half of the EPC with invasion patients showed hyperintensity on T1WI (P=0.19). A total of 63.6% of the EPC with invasion group showed non-mass enhancement surrounding (P=0.85). In addition, 29 patients (96.7%) had no axillary lymph node metastasis, and only one patient with EPC with invasion had axillary lymph node metastasis. Further pathological information analysis of EPCs showed that higher Ki-67 levels were more common in patients with EPCs with invasion (P=0.04). A total of 29 patients (96.7%) had the luminal phenotype, and one patient with EPC with invasion had the Her-2-positive phenotype. CONCLUSION: The margin, age and Ki-67 level were the key features for EPC risk stratification. In addition, these MRI signs, including a larger tumor, non-mass enhancement surrounding and axillary lymph node metastasis, may be suggestive of a high-risk stratification. Therefore, MRI phenotypes may provide additional information for the risk stratification of EPCs.

Effective apatinib treatment of pleomorphic liposarcoma: A case report.

RATIONALE: Pleomorphic liposarcoma (PLS) is a rare and aggressive malignant tumor, and both radiation and conventional cytotoxic chemotherapy remain controversial for metastatic or unresectable disease. PATIENT CONCERNS: We presented an 81-year-old Chinese woman with advanced PLS who received apatinib after failure chemotherapy. DIAGNOSES: The patient was diagnosed as having PLS by biopsy. INTERVENTIONS: After a failed chemotherapy, apatinib started to be taken orally 425 mg per day. OUTCOMES: This patient achieved 3-month progression-free survival (PFS) and a higher quality of life. Meanwhile, this patient suffered grade 2 hypertension and grade 3 hand-foot syndrome (HFS). LESSONS: In this case, apatinib presented good efficacy and safety to treat PLS. Randomized clinical studies are required to confirm the efficacy and safety of apatinib in the treatment of PLS.

[Effects of long-term and low-dose hormone replacement therapy on bone mineral density in postmenopausal women].

OBJECTIVE: To observe the effects of long-term and low-dose hormone replacement therapy on bone mineral density (BMD), and the incidence of bone pain in postmenopausal women. METHODS: Totally 141 postmenopausal women were selected from the medical staff of the Peking Union Medical College Hospital. Of them, 63 women treated with low-dose sex hormone for over 5 (5-32) years were divided into hormone replacement therapy (HRT) group, and 78 never receiving HRT were divided into control group. The BMD was measured by dual energy X-ray absorptiometry (DEXA) at lumbar spine, Ward's triangle, femoral neck, trochanter, and total hip, and the incidence of bone pain was inquired. RESULTS: The BMD in the HRT group was 9.1% higher than that in the control group (P < 0.05). The incidence of bone pain was significantly lower in the HRT group (71.4%) than that in the control group (89.7%). CONCLUSION: Long-term and low-dose hormone replacement therapy can reduce bone loss and the incidence of bone pain.

[Clinical characteristics of 17alpha-hydroxylase/17,20-lyase deficiency and long term follow up results].

OBJECTIVE: To improve the diagnosis and the treatment level of 17alpha-hydroxylase/17,20-lyase deficiency. METHODS: 24 patients suffering from this disease were diagnosed and treated in Peking Union Medical College Hospital from 1978 to 2002. All the patients had comprehensive hormone assay and some had bone mineral density detection. RESULTS: 20 patients with complete combined defect in this study had typical clinical presentation of hypertension and hypokalemia and presented both karyotype 46, XX or 46, XY. All had female external genitalia and lacked spontaneous puberty. The circulating concentrations of cortisol and gonadal steroids were reduced and that of gonadotropin was elevated, whereas in the remaining 4 cases with partial combined deficiencies, adrenal corticotropic hormone stimulating test as well as the results of the sex hormone determination suggested that there was partial 17alpha-hydroxylase/17,20-lyase enzyme activities in their adrenal glands or sexual glands. Long term follow up data showed that in most of the patients, small doses of dexamethasone (0.1 - 0.375 mg per day) could achieve good control of hypertension and correct hypokalemia, but in five patients other drugs were needed for normalization of blood pressure. With sex hormone replacement, the patients could keep adult female appearance, but did not have reproductive function. CONCLUSIONS: In clinical work it is necessary to enhance the recognition of partial combined 17alpha-hydroxylase/17,20-lyase deficiency. Long term regulation treatment can achieve good control of blood pressure and rectify the low blood potassium.

[Right attitude towards the postmenopausal hormone replacement therapy].

We have gone through decades using hormone replacement therapy (HRT). The first problem encountered was increased endometrial cancer and solved by addition of progesterone. Now we are facing cardiovascular complications and how could we solve in the use of HRT. Research in vitro with HUAR and HUVEC and clinically seemed to show that small physiological doses might be the solution in protection of CVD.

[Effects of sex hormones on serum levels of nitric oxide and plasma angiotensin II in postmenopausal women].

OBJECTIVE: To observe the effect of estrogen and progestin on the blood levels of nitric oxide and angiotensin II in aid of the application of hormone replacement therapy in postmenopausal women. METHODS: The serum nitric oxide and plasma angiotensin II levels in postmenopausal women were determined before and 3 months after oral intake of estradiol valerate 1 mg/day (n = 10) or estradiol valerate, 1 mg/d plus medroxyprogesterone acetate, 2 mg/d (n = 30). RESULTS: The serum nitric oxide levels of postmenopausal women were significantly increased by 3 months of oral estradiol valerate 1 mg/d (P < 0.05), whereas the plasma levels of angiotensin II tended to decrease. The positive correlation between the increases of nitric oxide and the changes of estradial 3 months after oral intake of estradiol valerate 1 mg/d was significant. Compared with the baseline, no significant changes were observed in both serum nitric oxide levels and plasma angiotensin II levels 3 months after oral intake of estradiol valerate, 1 mg/d plus medroxyprogesterone acetate, 2 mg/d (P < 0.05). CONCLUSIONS: The vascular functions can be improved through increasing the serum nitric oxide level after 3-month oral intake of estradiol valerate, 1 mg/d in postmenopausal women, and estradiol valerate plus medroxyprogesterone acetate intake may attenuate the beneficial effects.

[Clinical validity of anti-thyroperoxidase antibody and anti-thyroglobulin antibody].

OBJECTIVE: To evaluate the clinical validity of anti-thyroperoxidase antibody (anti-TPOAb) and anti-thyroglobulin antibody (anti-TgAb). METHOD: Serum levels of anti-TPOAb and anti-TgAb were assayed using chemiluminescence immunoassay in 434 subjects, including 51 patients with Hashimoto's thyroiditis, 58 with Graves' disease, 68 with nodular goiter, 56 with thyroid adenoma and carcinoma, 56 with subacute thyroiditis, 65 with euthyroid non-thyroid endocrine disease, 35 with euthyroid non-thyroid autoimmune diseases, and 45 euthyroid controls. RESULTS: The highest level and most positive results of serum anti-TgAb and anti-TPOAb were observed in patients with Hashimoto's thyroiditis (median 373 and 6 974 U/ml, positive rate 84.3% and 86.3%), followed by patients with Graves' disease (median 84 and 1 369 U/ml, positive rate 44.8% and 72.4%). Serum anti-TgAb and anti-TPOAb were also more common in patients with subacute thyroiditis and other autoimmune diseases than in the controls. CONCLUSION: The assay of serum anti-TPOAb and anti-TgAb by chemiluminescence immunoassy are useful in the differential diagnosis of autoimmune thyroid disease.

[Expression of transforming growth factor alpha, tumor necrosis factor alpha, and vascular endothelial growth factor of human pheochromocytoma tissues].

OBJECTIVE: To compare the expressions of transforming growth factor alpha (TGFalpha), tumor necrosis factor alpha (TNFalpha), and vascular endothelial growth factor (VEGF) between pheochromocytoma (PHEO) tissues and normal adrenal medulla tissues. METHODS: The mRNA expressions of TGFalpha, TNFalpha, and VEGF detected by RT-PCR, were compared between 22 PHEO tissues and 18 normal adrenal medulla tissues (according with the principle of medical ethnics). Immunohistochemistry staining was performed on 27 PHEO tissues and 14 normal adrenal medulla tissues. The comparisons of the protein expression of TGFalpha, TNFalpha, and VEGF were analyzed in both of PHEO tissues and normal adrenal medulla tissues. RESULTS: Compared with normal adrenal medulla tissues, the expressions of TGFalpha and TNFalpha mRNA and protein were higher in PHEO tissues, and VEGF145 mRNA expression was also higher in PHEO tissues, while there was no significant difference of the mRNA expression of VEGF121 and VEGF165 between these two tissues. Positive staining rates for VEGF of endothelial cells and tumor cells were higher in PHEO tissues than in normal adrenal medulla tissues. Expressions of the TGFalpha, TNFalpha, and VEGF protein were higher in extra-adrenal PHEO than in adrenal PHEO. The TNFalpha immunohistochemistry staining rate was higher in the malignant or multiple PHEO than in the benign or single PHEO. CONCLUSIONS: The mRNA and protein expressions of TGFalpha, TNFalpha, and VEGF are higher in PHEO tissues than those in normal adrenal medulla tissues. Expressions of these cytokines vary in PHEO with different characteristic.