Membrane-Located Expression of Thioesterase From Acinetobacter baylyi Enhances Free Fatty Acid Production With Decreased Toxicity in Synechocystis sp. PCC6803

It has been previously reported that photosynthetic production of extracellular free fatty acids (FFAs) in cyanobacteria was realized by thioesterases (TesA) mediated hydrolysis of fatty acyl-ACP in cytosol and excretion of the FFA outside of the cell. However, two major issues related to the genetically modified strains need to be addressed before the scale-up commercial application becomes possible: namely, the toxicity of FFAs, and the diversity of carbon lengths of fatty acids that could mimic the fossil fuel. To address those issues, we hypothesized that generating FFAs near membrane could facilitate rapid excretion of the FFA outside of the cell and thus decrease toxicity caused by intracellular FFAs in the cytosolic expression of thioesterase. To realize this, we localized a leaderless thioesterase (AcTesA) from Acinetobacter baylyi on the cytosolic side of the inner membrane of Synechocystis sp. PCC6803 using a membrane scaffolding system. The engineered strain with AcTesA on its membrane (mAcT) produced extracellular FFAs up to 171.9 ± 13.22 mg⋅L-1 compared with 40.24 ± 10.94 and 1.904 ± 0.158 mg⋅L-1 in the cytosol-expressed AcTesA (AcT) and wild-type (WT) strains, respectively. Moreover, the mAcT strain generated around 1.5 and 1.9 times less reactive oxygen species than AcT and WT, respectively. Approximately 78% of total FFAs were secreted with an average rate of 1 mg⋅L-1⋅h-1, which was higher than 0.44 mg⋅L-1⋅h-1 reported previously. In the case of mAcT strain, 60% of total secreted FFAs was monounsaturated (C18:1) which is the preferable biodiesel component. Therefore, the engineered mAcT strain shows enhanced FFAs production with less toxicity which is highly desirable for biodiesel production

Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics

GLP-1 (Glucagon-like peptide-1) is a peptide that stimulates insulin secretion from the β-cell for glycemic control of the plasma blood glucose level. Its mimetic exenatide (synthetic Exendin-4) with a longer half-life of approximately 3.3–4 h is widely used in clinical application to treat diabetes. Currently, exenatide is chemically synthesized. In this study, we report that the GLP-1 analogue recombinant Exendin-4 (Exdn-4) can be produced at a high level in Nicotiana benthamiana, with an estimated yield of 50.0 µg/g fresh biomass. For high-level expression, we generated a recombinant gene, B:GB1:ddCBD1m:8xHis : Exendin-4 (BGC : Exdn-4), for the production of Exendin-4 using various domains such as the BiP signal peptide, the GB1 domain (B1 domain of streptococcal G protein), a double cellulose binding domain 1 (CBD1), and 8 His residues (8xHis) to the N-terminus of Exendin-4. GB1 was used to increase the expression, whereas double CBD1 and 8xHis were included as affinity tags for easy purification using MCC beads and Ni(2+)-NTA resin, respectively. BGC : Exdn-4 was purified by single-step purification to near homogeneity using both Ni(2+)-NTA resin and microcrystalline cellulose (MCC) beads. Moreover, Exdn-4 without any extra residues was produced from BGC : Exdn-4 bound onto MCC beads by treating with enterokinase. Plant-produced Exdn-4 (Exendin-4) was as effective as chemically synthesized Exendin-4 in glucose-induced insulin secretion (GIIS) from mouse MIN6m9 cells a pancreatic beta cell line

Evaluation of the effect of aromatase inhibitor in reducing the size of endometrioma

Background: Endometriosis is a chronic and progressive estrogen-dependent disorder that can result in substantial morbidity, including pelvic pain, multiple operations, and infertility. Endometriosis can be ovarian, peritoneal or deep infiltrative. Blocking estrogen production by inhibiting aromatization, aromatase inhibitor (letrozole) has been shown to reduce the size of endometrioma and endometriosis associated pain. Aim of the study was to evaluate the effect of aromatase inhibitor in reducing the size of endometrioma.Methods: A prospective non comparative observational study was conducted in the Department of Reproductive Endocrinology and Infertility of BSMMU on 30 women with ovarian endometrioma during the period of April 2019 to March 2020. Women were treated with aromatase inhibitor (letrozole) 2.5 mg, norethisterone 5 mg, calcium 1200 mg, and vitamin D 800 IU daily for 6 months. Transvaginal ultrasound was performed at baseline, 3 months and 6 months after treatment to assess the mean diameter and volume of endometriomas. Statistical analyses were carried out by using the Statistical Package for Social Sciences version 23.0.Results: More than 50% reduction in volume occurred in 90% of endometrioma. In one (3.3%) case endometrioma disappeared completely after 6 months. There was statistically significant reduction of size of endometrioma (estimated by mean diameter and volume) and pain. Volume decrease was linearly related to baseline endometrioma volume and inversely related to baseline body mass index (BMI). The side effects were mild and well tolerated by the patients.Conclusions: Treatment of ovarian endometrioma with aromatase inhibitor combined with progestin add-back for 6 months cause substantial reduction in size of endometrioma and associated pain

Comparison of vitamin D (25OHD) status between fertile and infertile men

Background: Vitamin D (25OHD) deficiency has become a modern-day epidemic, being the most common nutritional deficiency worldwide. Many infertile men are experiencing low total sperm count or different semen abnormalities. The aim of this study was to compare serum vitamin D (25OHD) status among fertile and infertile men.Methods: This was an observational (cross sectional comparative) study and was conducted in the Department of Reproductive Endocrinology and Infertility, BSMMU, Dhaka, Bangladesh during the period from April 2019 to March 2020. The sample size was 112 men where 56 participants were in fertile men group and 56 participants were infertile men group. Statistical analyses were carried out by using Windows based Statistical Package for Social Sciences (SPSS, version 23.0).Results: The predictability of vitamin D insufficiency was significant. Holding the effects of vitamin D deficiency constant, males with vitamin D insufficiency were 3.28 times more likely to be infertile than males with vitamin D sufficiency. Subgroup analysis of infertile men was done regarding semen parameters in different vitamin D status categories. There was statistically significant difference in semen volume and sperm concentration between infertile men of different vitamin D status but no significant difference in case of motility and morphology.Conclusions: There was no significant different of serum vitamin D (25OHD) between fertile and infertile men. Men with vitamin D insufficiency (≥20 ng/ml to <30 ng/ml) are more likely to be infertile than men with vitamin D sufficiency.

In vitro antibacterial properties of essential oil and organic extracts of Premna integrifolia Linn

The aims of this study were to examine the chemical composition of the essential oil of Premna integrifolia Linn (Lamiaceae), and to test the efficacy of the oil and various organic extracts as an antibacterial potential. The chemical compositions of the essential oil were analyzed by GC–MS. Twenty-nine compounds representing 94.81% of the total leaves oil were identified, of which phytol (27.25%), α-humulene (14.21%), spathulenol (12.12%), 1-octen-3-ol (8.21%), eugenol (6.69%) and phenylethyl alcohol (5.81%) were the major compounds. The oil (15 μL disk−1) and extracts (300 μg disk−1) of P. integrifolia displayed a great potential of antibacterial activity against Sarcina lutea IFO 3232, Bacillus subtilis IFO 3026, Escherichia coli IFO 3007, Pseudomonas sp. ATCC 13867, Klebsiella pneumoniae ATCC 10031 and Xanthomonas campestries IAM 1671 with their respective zones of inhibition of 12.0 ± 1.2 to 22.1 ± 1.2 mm and MIC values of 62.5–250 μg mL−1. The results of this study suggest that the natural products derived from P. integrifolia may have potential use in food, pharmaceutical and/or agro industries for preservatives or antimicrobial agents

Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics

GLP-1 (Glucagon-like peptide-1) is a peptide that stimulates insulin secretion from the β-cell for glycemic control of the plasma blood glucose level. Its mimetic exenatide (synthetic Exendin-4) with a longer half-life of approximately 3.3–4 h is widely used in clinical application to treat diabetes. Currently, exenatide is chemically synthesized. In this study, we report that the GLP-1 analogue recombinant Exendin-4 (Exdn-4) can be produced at a high level in Nicotiana benthamiana, with an estimated yield of 50.0 µg/g fresh biomass. For high-level expression, we generated a recombinant gene, B:GB1:ddCBD1m:8xHis : Exendin-4 (BGC : Exdn-4), for the production of Exendin-4 using various domains such as the BiP signal peptide, the GB1 domain (B1 domain of streptococcal G protein), a double cellulose binding domain 1 (CBD1), and 8 His residues (8xHis) to the N-terminus of Exendin-4. GB1 was used to increase the expression, whereas double CBD1 and 8xHis were included as affinity tags for easy purification using MCC beads and Ni2+-NTA resin, respectively. BGC : Exdn-4 was purified by single-step purification to near homogeneity using both Ni2+-NTA resin and microcrystalline cellulose (MCC) beads. Moreover, Exdn-4 without any extra residues was produced from BGC : Exdn-4 bound onto MCC beads by treating with enterokinase. Plant-produced Exdn-4 (Exendin-4) was as effective as chemically synthesized Exendin-4 in glucose-induced insulin secretion (GIIS) from mouse MIN6m9 cells a pancreatic beta cell line.11Ysciescopu