Production of nanocellulose in miniature-bioreactor: Optimization and characterization

Bacterial cellulose (BC) is a very fascinating microbial biopolymer which is mainly produced by Gluconacetobacter xylinum. Optimization of BC production by G. xylinum was performed based on scale-down studies in miniature-bioreactor and response surface methodology in which the optimum pH value (6.5) and shaking rate (50?rpm) were obtained. The static culture condition for BC production has newly been defined. Nanostructure of BC includes nanofibers up to (60?nm) and nanoporosity up to (265?nm) was observed by scanning electron microscopy. By Fourier transform infrared spectroscopy study, the most expected BC interaction is nucleophilic interaction. MTT assay showed high biocompatibility. Appropriate mechanical strength (0.37?MPa) and Young’s modulus (3.36?MPa) evinced BC scaffold utilization for skin tissue. The results indicate that BC sheets can be utilized in biomedical application and nanotechnology approaches.     

Long bone mesenchymal stem cells (Lb-MSCs): clinically reliable cells for osteo-diseases

Mesenchymal stem cells (MSCs) have been designated as the most reliable cells in clinics to treat osteo-diseases because of their versatile nature. MSCs, isolated from long bone (Lb-MSCs) are rarely reported and named as RIA-MSCs because of the reamer–irrigator–aspirator (RIA) device. The potential of these cells in the treatment of non-union bone fractures made them the ideal candidates to be studied for clinical practices. In this work, effect of cryopreservation on the proliferation and differentiation capabilities of long bone MSCs (Lb-MSCs) has been studied. For this purpose, Lb-MSCs were isolated via RIA device and characterized using flow cytometry and differentiation assays. Cells were cryopreserved for 3, 6 and 12 months and thereafter were characterized using differentiation assays and genetic markers specific for osteogenic, chondrogenic, and adipogenic potential quantitatively by qRT-PCR. Lb-MSCs were found expressing MSC characteristic markers defining their identity. The population doubling time (PDT) was about 2.5 ± 0.5 days and colonies appeared after 7–10 days. Differentiation potential and gene expression of 3, 6 and 12 months cryopreserved Lb-MSCs were unaltered. The results show that cryopreservation did not have an effect on the differentiation potential of human Lb-MSCs. Therefore, our work offers Lb-MSCs as clinically cells for treating osteo-diseases.     

Noninvasive Fetal Sex Determination by Real-Time PCR and TaqMan Probes

Background:Noninvasive fetal sex determination by analyzing Y chromosome-specific sequences is very useful in the management of cases related to sex-linked genetic diseases. The aim of this study was to establish a non-invasive fetal sex determination test using Real-Time PCR and specific probes.Methods:The study was a prospective observational cohort study conducted from August 2018 to September 2019. Venous blood samples were collected from 25 Iranian pregnant women at weeks 7 to 25 of gestation. Cell-free DNA (cfDNA) was isolated from the plasma of samples and fetal sex was determined by SRY gene analysis using the Real-Time PCR technique. In the absence of SRY detection, the presence of fetal DNA was investigated using cfDNA treated with BstUI enzyme and PCR for the epigenetic marker RASSF1A.Results:Of the total samples analyzed, 48% were male and 52% female. The RASSF1A assay performed on SRY negative cases also confirmed the presence of cell-free fetal DNA. Genotype results were in full agreement with neonate gender, and the accuracy of noninvasive fetal sex determination was 100%.Conclusion:Fetal sex determination using the strategy applied in this study is noninvasive and highly accurate and can be exploited in the management of sex-linked genetic diseases.Key Words: Cell-free fetal DNA, Fetal sex determination, Noninvasive prenatal diagnosis, Sex-linked genetic diseases, SRY     

Towards a global perspective for Salvia L.: Phylogeny,diversification and floral evolution

Salvia is the most species-rich genus in Lamiaceae, encompassing approximately 1000 species distributed all over the world. We sought a new evolutionary perspective for Salvia by employing macroevolutionary analyses to address the tempo and mode of diversification. To study the association of floral traits with speciation and extinction, we modelled and explored the evolution of corolla length and the lever-mechanism pollination system across our Salvia phylogeny. We reconstructed a multigene phylogeny for 366 species of Salvia in the broad sense including all major recognized lineages and 50 species from Iran, a region previously overlooked in studies of the genus. Our comprehensive sampling of Iranian species of Salvia provides higher phylogenetic resolution for southwestern Asian species than obtained in previous studies. Our phylogenetic data in combination with divergence time estimates were used to examine the evolution of corolla length, woody versus herbaceous habit, and presence versus absence of a lever mechanism. We investigated the timing and dependence of Salvia diversification related to corolla length evolution through a disparity test and BAMM analysis. A HiSSE model was used to evaluate the dependency of diversification on the lever-mechanism pollination system in Salvia. A medium corolla length (15–18 mm) was reconstructed as the ancestral state for Salvia with multiple shifts to shorter and longer corollas. Macroevolutionary model analyses indicate that corolla length disparity is high throughout Salvia evolution, significantly different from expectations under a Brownian motion model during the last 28 million years of evolution. Our analyses show evidence of a higher diversification rate of corolla length for some Andean species of Salvia compared to other members of the genus. Based on our tests of diversification models, we reject the hypothesis of a direct effect of the lever mechanism on Salvia diversification. Therefore, we suggest caution in considering the lever-mechanism pollination system as one of the main drivers of speciation in Salvia.     

Evaluating the mechanism underlying antitumor effect of interleukin 27 on B cells of chronic lymphocytic leukemia patients

Chronic lymphocyte leukemia (CLL) is a B-cell malignancy resisted to apoptosis. Recently, some studies indicated that cytokines such as interleukin 27 (IL-27) can reduce B-cell proliferation. The aim of this study is to evaluate the mechanism underlying the proapoptotic effect of IL-27 on B cells of patients with CLL in comparison with B cells of normal subjects. The effect of IL-27 on the antitumor activity of natural killer (NK) and T cells was also evaluated. Peripheral blood mononuclear cells (PBMCs) were isolated from 35 patients with CLL and 15 normal subjects. B cells and PBMCs were cocultured with IL-27 and B cells apoptosis to evaluate proliferation. Both messenger RNA and protein expression of IL-27 and IL-27 receptor were determined using flow cytometry and real-time polymerase chain reaction analysis. To evaluate the apoptotic effect of IL-27 on B cells of patients with CLL, Annexin V-FITC and 7-AAD (BioLegend) fluorescent dyes were used. In addition, the IL-27 effect on activation of T cell and NK cell was determined by determining CD96 molecule expression. IL-27 and IL-27 receptor expression in patients with CLL was significantly lower than that of normal subjects (p < .05). IL-27 enhanced apoptosis of B cells in patients with CLL (p < .05) but this effect was not significantly observed in B cells of normal subjects (p > .05). Consequently, IL-27 reduced the proliferation of B cells and enhanced NK cell activity (p < .05). IL-27, through inducing apoptosis, can exert an inhibitory effect on cancer B cells of CLL patients with minimal effect on normal B cells.     

Exploiting yoeB-yefM toxin-antitoxin system of Streptococcus pneumoniae on the selective killing of miR-21 overexpressing breast cancer cell line (MCF-7)

Toxin-antitoxin (TA) systems are two-component genetic modules widespread in bacterial and archaeal genomes, in which the toxin module is rendered inactive under resting conditions by its antitoxin counterpart. Under stress conditions, however, the antitoxin is degraded, freeing the toxin to exert its lethal effects. Although not evolved to function in eukaryotes, some studies have established the lethal activity of these bacterial toxins by inducing apoptosis in mammalian cells, an effect that can be neutralized by its cognate antitoxin. Inspired by the way the toxin can become active in eukaryotes cells, we produced an engrained yoeB-yefM TA system to selectively kill human breast cancer cells expressing a high level of miR-21. Accordingly, we generated an engineered yefM antitoxin gene with eight miR-21 target sites placed in its 3′untranslated region. The resulting TA system acts autonomously in human cells, distinguishing those that overexpress miR-21, killed by YoeB, from those that do not, remaining protected by YefM. Thus, we indicated that microRNA-control of the antitoxin protein of bacterial TA systems constitutes a novel strategy to enhance the selective killing of human cancer cells by the toxin module. The present study provides significant insights for developing novel anticancer strategies avoiding off-target effects, a challenge that has been pursued by many investigators over the years.     

Targeted Anticancer Drug Delivery Using Chitosan,Carbon Quantum Dots,and Aptamers to Deliver Ganoderic Acid and 5-Fluorouracil

Breast cancer is a malignancy that affects mostly females and is among the most lethal types of cancer. The ligand-functionalized nanoparticles used in the nano-drug delivery system offer enormous potential for cancer treatments. This work devised a promising approach to increase drug loading efficacy and produce sustained release of 5-fluorouracil (5-FU) and Ganoderic acid (GA) as model drugs for breast cancer. Chitosan, aptamer, and carbon quantum dot (CS/Apt/COQ) hydrogels were initially synthesized as a pH-sensitive and biocompatible delivery system. Then, CS/Apt/COQ NPs loaded with 5-FU-GA were made using the W/O/W emulsification method. FT-IR, XRD, DLS, zeta potentiometer, and SEM were used to analyze NP's chemical structure, particle size, and shape. Cell viability was measured using MTT assays in vitro using the MCF-7 cell lines. Real-time PCR measured cell apoptotic gene expression. XRD and FT-IR investigations validated nanocarrier production and revealed their crystalline structure and molecular interactions. DLS showed that nanocarriers include NPs with an average size of 250.6 nm and PDI of 0.057. SEM showed their spherical form, and zeta potential studies showed an average surface charge of +37.8 mV. pH 5.4 had a highly effective and prolonged drug release profile, releasing virtually all 5-FU and GA in 48 h. Entrapment efficiency percentages for 5-FU and GA were 84.7±5.2 and 80.2 %±2.3, respectively. The 5-FU-GA-CS-CQD-Apt group induced the highest cell death, with just 57.9 % of the MCF-7 cells surviving following treatment. 5-FU and GA in CS-CQD-Apt enhanced apoptotic induction by flow cytometry. 5-FU-GA-CS-CQD-Apt also elevated Caspase 9 and downregulated Bcl2. Accordingly, the produced NPs may serve as pH-sensitive nano vehicles for the controlled release of 5-FU and GA in treating breast cancer.     

Label-Free LSPR Prostate-Specific Antigen Immune-Sensor Based on GLAD-Fabricated Silver Nano-columns

Label-free detection of biomarkers has been recently noticed and optical biosensors showed great potential to be the method of choice in such situation. Here, we used glancing angle deposition (GLAD) method in which silver nano-columns stabilized by a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) and 6-mercaptohexanol to investigate the capability of localized surface plasmon resonance (LSPR)–based silver nanochips to detect prostate-specific antigen (PSA). Using different standard solutions of PSA, limit of detection (LOD) of the nano-sensors has been calculated to be 850 pg/ml. The selectivity of the nano-sensors has also been evaluated. We showed that these nano-sensors could detect PSA in clinically acceptable sensitivity and specificity without any complicated laboratory equipment.

     

Preparation and Characterization of Zein/Sodium Caseinate/Xanthan Gum Complex for Encapsulation of Piperine and its In Vitro Release Study

To encapsulate piperine (Pip), as a poor water-soluble bioactive compound, zein-sodium caseinate-xanthan gum (Z-SG-XG) nanocomplex was prepared as a colloidal delivery system. The effect of different parameters involved in complexation process, including concentration of proteins, polysaccharide, and Pip on the encapsulation efficiency of Pip, particle size and stability of the nanocomplexes was investigated. Powders obtained by freeze-drying of the colloidal solution had relatively uniform particles compared to those obtained from conventional drying system and showed well redispersibility in water. At the optimal condition, a stable and homogeneous nanocomplex with a mean particle size of 145.9 ± 2.7 nm, PDI of 0.27 ± 0.01, and ζ-potential of −39.7 ± 1.3 mV was obtained. The antioxidant activity of Pip was significantly improved by encapsulation into the Z-SC-XG nanocomplex. Also, the in vitro release of Pip from the synthesized nanocomplexes in phosphate-buffer saline (PBS) solution and simulated gastrointestinal fluids (SGIF) was investigated and the release kinetic was studied as well. The Pip/Z-SG-XG nanocomplex showed a slower release in SGIF compared to the free Pip and nanoparticles without XG and SC, while its antioxidant activity was remarkable. Results suggested a possible utilization of Z-SC-XG nanocomplex for improving the water solubility, bioavailability and storage stability of Pip.