Polymeric nanoparticles (PNPs) for oral delivery of insulin.

Successful oral insulin administration can considerably enhance the quality of life (QOL) of diabetes patients who must frequently take insulin injections. Oral insulin administration, on the other hand, is seriously hampered by gastrointestinal enzymes, wide pH range, mucus and mucosal layers, which limit insulin oral bioavailability to ≤ 2%. Therefore, a large number of technological solutions have been proposed to increase the oral bioavailability of insulin, in which polymeric nanoparticles (PNPs) are highly promising for oral insulin delivery. The recently published research articles chosen for this review are based on applications of PNPs with strong future potential in oral insulin delivery, and do not cover all related work. In this review, we will summarize the controlled release mechanisms of oral insulin delivery, latest oral insulin delivery applications of PNPs nanocarrier, challenges and prospect. This review will serve as a guide to the future investigators who wish to engineer and study PNPs as oral insulin delivery systems.

Effects of Age and Biological Age-Determining Factors on Telomere Length in Type 2 Diabetes Mellitus Patients.

Background and Objectives: Telomere length (TL) undergoes attrition over time, indicating the process of aging, and is linked to a higher risk of diabetes mellitus type 2 (DM-2). This molecular epidemiological study investigated the correlation between leukocyte TL variations and determinants of molecular aging in 121 Pakistani DM-2 patients. Materials and Methods: The ratio of telomere repeats to the SCG copy number was calculated to estimate the TL in each sample through qPCR assays. Results: In this study, smaller mean TLs were observed in 48.8% of males (6.35 ± 0.82 kb), 3.3% of underweight patients (5.77 ± 1.14 kb), 61.2% of patients on regular medication (6.50 ± 0.79 kb), 9.1% with very high stress levels (5.94 ± 0.99 kb), 31.4% of smokers (5.83 ± 0.73 kb), 40.5% of patients with low physical activity (6.47 ± 0.69 kb), 47.9% of hypertensive patients (5.93 ± 0.64 kb), 10.7% of patients with DM-2 for more than 15 years, and 3.3% of patients with a delayed onset of DM-2 (6.00 ± 0.93 kb). Conclusion: This research indicated a significant negative correlation (R2 = 0.143) between TL and the age of DM-2 patients. This study demonstrated that the correlation of telomere length with age in DM-2 patients was also influenced by various age-determining factors, including hypertension and smoking habits, with significant strong (R2 = 0.526) and moderate (R2 = 0.299) correlations, respectively; sex, obesity, the stress level and age at the onset of diabetes with significant weak correlations (R2 = 0.043, 0.041, 0.037, and 0.065, respectively), and no significant correlations of medication routine, rate of physical activity, and the durations of DM-2 with age-adjusted telomere length. These results challenge TL as the sole marker of aging, thus highlighting the need for further research to understand underlying factors and mitigate the effect of aging or premature aging on diabetic patients.

Isolation, Identification and Characterization of Growth Parameters of Pseudomonas putida HSM-C2 with Coumarin-Degrading Bacteria.

Natural coumarins contribute to the aroma of licorice, and they are often used as a flavoring and stabilizing agents. However, coumarins usage in food has been banned by various countries due to its toxic effect. In this study, a strain of HSM-C2 that can biodegrade coumarin with high efficiency was isolated from soil and identified as Pseudomonas putida through performing 16S rDNA sequence analysis. The HSM-C2 catalyzed the biodegradation up to 99.83% of 1 mg/mL coumarin within 24 h under optimal culture conditions, such as 30 °C and pH 7, which highlights the strong coumarin biodegrading potential of this strain. The product, such as dihydrocoumarin, generated after the biodegradation of coumarin was identified by performing GC-MS analysis. The present study provides a theoretical basis and microbial resource for further research on coumarin biodegradation.

Biogenic Synthesis of Silver Nanoparticles Using Catharanthus roseus and Its Cytotoxicity Effect on Vero Cell Lines.

Background: Type 2 diabetes mellitus (DM2) is a chronic and sometimes fatal condition which affects people all over the world. Nanotherapeutics have shown tremendous potential to combat chronic diseases­including DM2­as they enhance the overall impact of drugs on biological systems. Greenly synthesized silver nanoparticles (AgNPs) from Catharanthus roseus methanolic extract (C. AgNPs) were examined primarily for their cytotoxic and antidiabetic effects. Methods: Characterization of C. AgNPs was performed by UV−vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and atomic force microscopy (AFM). The C. AgNPs were trialed on Vero cell line and afterwards on an animal model (rats). Results: The C. AgNPs showed standard structural and functional characterization as revealed by FTIR and XRD analyses. The zetapotential analysis indicated stability while EDX analysis confirmed the formation of composite capping with Ag metal. The cytotoxic effect (IC50) of C. AgNPs on Vero cell lines was found to be 568 g/mL. The animal model analyses further revealed a significant difference in water intake, food intake, body weight, urine volume, and urine sugar of tested rats after treatment with aqueous extract of C. AgNPs. Moreover, five groups of rats including control and diabetic groups (NC1, PC2, DG1, DG2, and DG3) were investigated for their blood glucose and glycemic control analysis. Conclusions: The C. AgNPs exhibited positive potential on the Vero cell line as well as on experimental rats. The lipid profile in all the diabetic groups (DG1-3) were significantly increased compared with both of the control groups (p < 0.05). The present study revealed the significance of C. AgNPs in nanotherapeutics.

Engineering Saccharomyces cerevisiae for high yield production of α-amyrin via synergistic remodeling of α-amyrin synthase and expanding the storage pool.

α-Amyrin is a plant-originated high-valued triterpene that is highly effective against several pathological ailments. α-Amyrin production by engineered Saccharomyces cerevisiae has been achieved by introducing α-amyrin synthase (αAS). However, the low yield of α-amyrin highly limits its industrial application; the low catalytic activity of αAS and the toxic effect of α-amyrin have been considered key elements. In this study, the highest yield of α-amyrin was obtained in engineered S. cerevisiae by remodeling α-amyrin synthase MdOSC1 and expanding the storage pool. The yield of α-amyrin was increased to 11-fold higher than that of the control by the triple mutant MdOSC1N11T/P250H/P373A obtained based on the modeling analysis. Furthermore, key genes of MVA pathway were overexpressed to provide sufficient precursors, and DGA1 (Diacylglycerol acyltransferase) was overexpressed to expand the intracellular storage capacity. Finally, the as-constructed aAM12 strain produced 213.7 ± 12.4 mg/L α-amyrin in the shake flask and 1107.9 ± 76.8 mg/L in fed-batch fermentation; the fermentation yield was 106-fold higher than that of the original aAM1 strain under the same conditions, representing the highest α-amyrin yield in yeast reported to date. Microbial production of α-amyrin with over 1 g/L will be suitable for commercialization and can accelerate the industrial production of α-amyrin in yeast.

Synergistic Neuroprotective Effect of Endogenously-Produced Hydroxytyrosol and Synaptic Vesicle Proteins on Pheochromocytoma Cell Line Against Salsolinol.

Oxidative stress triggers a lethal cascade, leading to Parkinson's disease by causing degeneration of dopaminergic neurons. In this study, eight antioxidants were screened for their neuroprotective effect on PC12 cells (pheochromocytoma cell line) under oxidative stress induced by salsolinol (OSibS). Hydroxytyrosol was found to be the strongest neuroprotective agent; it improved viability of PC12 cells by up to 81.69% under OSibS. Afterward, two synaptic vesicle proteins, synapsin-1 and septin-5, were screened for their neuroprotective role; the overexpression of synapsin-1 and the downregulation of septin-5 separately improved the viability of PC12 cells by up to 71.17% and 67.00%, respectively, compared to PC12 cells only treated with salsolinol (PoTwS) under OSibS. Subsequently, the PC12+syn++sep- cell line was constructed and pretreated with 100 µM hydroxytyrosol, which improved its cell viability by up to 99.03% and led to 14.71- and 6.37-fold reductions in the levels of MDA and H2O2, respectively, and 6.8-, 12.97-, 10.57-, and 7.57-fold increases in the activity of catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase, respectively, compared to PoTwS under OSibS. Finally, alcohol dehydrogenase-6 from Saccharomyces cerevisiae was expressed in PC12+syn++sep- cells to convert 3,4-dihydroxyphenylacetaldehyde (an endogenous neurotoxin) into hydroxytyrosol. The PC12+syn++sep-+ADH6+ cell line also led to 22.38- and 12.33-fold decreases in the production of MDA and H2O2, respectively, and 7.15-, 13.93-, 12.08-, and 8.11-fold improvements in the activity of catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase, respectively, compared to PoTwS under OSibS. Herein, we report the endogenous production of a powerful antioxidant, hydroxytyrosol, from 3,4-dihydroxyphenylacetaldehyde, and evaluate its synergistic neuroprotective effect, along with synapsin-1 and septin-5, on PC12 cells under OSibS.

Biosynthesis of Polyhydroxyalkanoates (PHAs) by the Valorization of Biomass and Synthetic Waste.

Synthetic pollutants are a looming threat to the entire ecosystem, including wildlife, the environment, and human health. Polyhydroxyalkanoates (PHAs) are natural biodegradable microbial polymers with a promising potential to replace synthetic plastics. This research is focused on devising a sustainable approach to produce PHAs by a new microbial strain using untreated synthetic plastics and lignocellulosic biomass. For experiments, 47 soil samples and 18 effluent samples were collected from various areas of Punjab, Pakistan. The samples were primarily screened for PHA detection on agar medium containing Nile blue A stain. The PHA positive bacterial isolates showed prominent orange-yellow fluorescence on irradiation with UV light. They were further screened for PHA estimation by submerged fermentation in the culture broth. Bacterial isolate 16a produced maximum PHA and was identified by 16S rRNA sequencing. It was identified as Stenotrophomonas maltophilia HA-16 (MN240936), reported first time for PHA production. Basic fermentation parameters, such as incubation time, temperature, and pH were optimized for PHA production. Wood chips, cardboard cutouts, plastic bottle cutouts, shredded polystyrene cups, and plastic bags were optimized as alternative sustainable carbon sources for the production of PHAs. A vital finding of this study was the yield obtained by using plastic bags, i.e., 68.24 ± 0.27%. The effective use of plastic and lignocellulosic waste in the cultivation medium for the microbial production of PHA by a novel bacterial strain is discussed in the current study.

A Novel ß-Glucuronidase from Talaromyces pinophilus Li-93 Precisely Hydrolyzes Glycyrrhizin into Glycyrrhetinic Acid 3-O-Mono-ß-d-Glucuronide.

Glycyrrhetinic acid 3-O-mono-ß-d-glucuronide (GAMG), which possesses a higher sweetness and stronger pharmacological activity than those of glycyrrhizin (GL), can be obtained by removal of the distal glucuronic acid (GlcA) from GL. In this study, we isolated a ß-glucuronidase (TpGUS79A) from the filamentous fungus Talaromyces pinophilus Li-93 that can specifically and precisely convert GL to GAMG without the formation of the by-product glycyrrhetinic acid (GA) from the further hydrolysis of GAMG. First, TpGUS79A was purified and identified through matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF-TOF MS) and deglycosylation, indicating that TpGUS79A is a highly N-glycosylated monomeric protein with a molecular mass of around 85 kDa, including around 25 kDa of glycan moiety. The gene for TpGUS79A was then cloned and verified by heterologous expression in Pichia pastoris TpGUS79A belonged to glycoside hydrolase family 79 (GH79) but shared low amino acid sequence identity (<35%) with the available GH79 GUS enzymes. TpGUS79A had strict specificity toward the glycan moiety but poor specificity toward the aglycone moiety. Interestingly, TpGUS79A recognized and hydrolyzed the distal glucuronic bond of GL but could not cleave the glucuronic bond in GAMG. TpGUS79A showed a much higher catalytic efficiency on GL (kcat/Km of 11.14 mM-1 s-1) than on the artificial substrate pNP ß-glucopyranosiduronic acid (kcat/Km of 0.01 mM-1 s-1), which is different from the case for most GUSs. Homology modeling, substrate docking, and sequence alignment were employed to identify the key residues for substrate recognition. Finally, a fed-batch fermentation in a 150-liter fermentor was established to prepare GAMG through GL hydrolysis by T. pinophilus Li-93. Therefore, TpGUS79A is potentially a powerful biocatalyst for environmentally friendly and cost-effective production of GAMG.IMPORTANCE Compared to chemical methods, the biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid 3-O-mono-ß-d-glucuronide (GAMG), which has a higher sweetness and stronger pharmacological activity than those of GL, via catalysis by ß-glucuronidase is an environmentally friendly approach due to the mild reaction conditions and the high yield of GAMG. However, currently available GUSs show low substrate specificity toward GL and further hydrolyze GAMG to glycyrrhetinic acid (GA) as a by-product, increasing the difficulty of subsequent separation and purification. In the present study, we succeeded in isolating a novel ß-glucuronidase (named TpGUS79A) from Talaromyces pinophilus Li-93 that specifically hydrolyzes GL to GAMG without the formation of GA. TpGUS79A also shows higher activity on GL than those of the previously characterized GUSs. Moreover, the gene for TpGUS79A was cloned and its function verified by heterologous expression in P. pastoris Therefore, TpGUS79A can serve as a powerful biocatalyst for the cost-effective production of GAMG through GL transformation.

Unravel the Supremacy of Klebsiella variicola over Native Microbial Strains for Aroma-Enhancing Compound Production in Reconstituted Tobacco Concentrate through Metagenomic Analysis.

Sensory attributes strongly influence consumers' preferences for products. The inoculation of the Klebsiella variicola H8 strain in a reconstituted tobacco leaf concentrate (RTLC) solution increased neutral aroma-enhancing compound (NAEC) production by 45%, decreased the nicotine level by 25%, decreased the water-soluble total sugar content by ~36%, and improved the sensory quality by 5.71%. The production of NAECs such as dihydrokiwi lactone (DHKL: 192.86%), 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene (THTMN: 177.77%), 2,4-di-tert-butylphenol (DTBP: 25%), 4-oxoisofolkone (OIFK: 116.66%,) 1,9-heptadecadiene-4,6-diyn-3-ol (HDD: 116.67%), ß-damastrone (BDS: 116.67), and megastigmatrienone A (MSTA: 116.67%) was increased. A metagenomics analysis of the microbial community in the fermented RTLC (FRTLC) was performed to elucidate the mechanism by which NAECs were produced. As a result, 24 groups of functional genes were identified, and among them, five families of carbohydrate-active enzymes, (i) glycoside hydrolase (GH), (ii) glycosyltransferase (GT), (iii) polysaccharide lyase (PL), (iv) carbohydrate esterase (CE), and (v) auxiliary active enzyme (AA), were found to be positively correlated with the production of NAECs. However, among the GHs, the GHs annotated from the H8 strain chromosome displayed the highest relative abundance and a positive correlation with the production of NAECs. Specifically, the GH13-14, GH13-20, GH13-38, GH13-25, GH13-10, GH42, and GH28 genes of the H8 strain were relatively more abundant and were key contributors to the production of NAECs. The correlation analyses revealed that the H8 strain plays a leading role among all the microorganisms in FRTLC in the production of NAECs. Our findings support the application of Klebsiella variicola in NAEC production and a reduction in nicotine content in tobacco products.

Arterial vascular cell line expressing SSAO: a new tool to study the pathophysiology of vascular amine oxidases.

Semicarbazide-sensitive amine oxidase (SSAO) widely exists in nature, mainly expressed at significant levels in vasculature. It plays a detrimental role in vascular diseases, particularly atherosclerosis, which occurs mainly in arteries. Herein we for the first time present SSAO expression in arterial lineage of vascular cell line, i.e., human umbilical arterial endothelial cell (HUAEC). Firstly, two commercially available gene transfection reagents were compared to determine high transfection efficiency and then the expression behavior of HUAEC:SSAO was characterized. Furthermore, our model was also been compared with commonly used human embryonic kidney (HEK) cell transfected with the same vector. For enzymatic assay, an in-house developed highly sensitive high performance liquid chromatography electron spray ionization mass spectrometry method was applied. Results indicated that the maximal transfection efficiency in HUAEC was detected by JetPEI™ and transfected protein was expressed at membrane and cytosol of different clones. No significant variations were observed in HUAEC between cell passages 1 and 7, although HEK cell displayed twofold higher SSAO expression level than HUAEC. The transfected SSAO was shown to be released into the cell-culture medium. Both cellular and released types of SSAO exhibited monomer and dimer structural forms. The cytotoxicity determination exhibited large number of viable cells after transfection with JetPEI™. Differential expression characterization of this new cell line demonstrates the correct behavior of SSAO in arterial endothelial cells and also provides a real physiological environment to elucidate the unclear role of this enzyme. In addition, our cellular model could partly solve the problems raised by the loss of enzyme expression found in cultured endothelial cells. This model could also be a useful tool for proteomic base study, screening of interacting protein and analysis of compounds that could modify its activity for therapeutic purposes.