Dental stem cell banking: Techniques and protocols

Dental tissue-derived stem cells (DSCs) provide an easy, accessible, relatively noninvasive promising source of adult stem cells (ASCs), which brought encouraging prospective for their clinical applications. DSCs provide a perfect opportunity to apply for a patient's own ASC, which poses a low risk of immune rejection. However, problems associated with the long-term culture of stem cells, including loss of proliferation and differentiation capacities, senescence, genetic instability, and the possibility of microbial contamination, make cell banking necessary. With the rapid development of advanced cryopreservation technology, various international DSC banks have been established for both research and clinical applications around the world. However, few studies have been published that provide step-by-step guidance on DSCs isolation and banking methods. The purpose of this review is to present protocols and technical details for all steps of cryopreserved DSCs, from donor selection, isolation, cryopreservation, to characterization and quality control. Here, the emphasis is on presenting practical principles in accordance with the available valid guidelines.     

The codon-optimization of cfaE gene and evaluating its high expression capacity and conserved immunogenicity in Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of children diarrhea in the world. Adhesion of ETEC to small intestine is an important virulence trait. One of the most prevalent colonization factors (CFs) in human is CFA/I fimbriae and CfaE which is the required binding factor for adhesion of ETEC to intestinal mucosa.We optimized cfaE gene codons according to codon bias of E. coli to achieve a high level of recombinant protein expression. The optimized gene was expressed in E. coli and rCFaE protein was used for mice immunization. Blocking activity of the obtained antibody was examined by microplate agglutination inhibition test. SDS-PAGE analysis indicated that the optimized sequence of cfaE produces a suitable amount of rCFaE in comparison with native gene sequence. This optimized rCFaE protein could induces strong humoral response in mice and the antibody obtained against rCFaE inhibited the adhesion of ETEC to human group A erythrocytes. It is concluded that codon optimization is a useful approach for obtaining large quantities of recombinant rCFaE protein. With regard to the results of hemagglutination inhibition test, codon optimization and increased production of recombinant protein expressed in E. coli did not affect the immunogenicity potential of CFaE.     

How the Nucleus and Mitochondria Communicate in Energy Production During Stress: Nuclear MtATP6, an Early-Stress Responsive Gene, Regulates the Mitochondrial F1F0-ATP Synthase Complex

A small number of stress-responsive genes, such as those of the mitochondrial F₁F₀-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F₀ part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F₁F₀-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.     

Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography

Abstract

The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400?SEC, we obtained highly pure, i.e.,?>?90%, rHBsAg virus-like particles (VLP).     

When to estimate sex ratio in natural populations of insects? A study on sex ratio variations of gall midges within a generation

Precise estimation of arthropods' sex ratio is an important issue in a wide range of ecological studies and biological control programs. Although, in many cases changes in arthropods' sex ratio may be under the control of parents or some symbiotic microorganisms, biased sex ratios in some other species are caused by some extrinsic factors, neglect of which may lead to under/overestimation of true sex ratio. In this paper, we pursued those factors that cause false estimation of sex ratio in insects' species. We studied the predatory gall midge, Aphidoletes aphidimyza Rondani (Diptera: Cecidomyiidae), an important biological control agent of aphids, that shows protandry (i.e. early male emergence), differential lifespan of sexes, and differential distribution of sexes across habitat. Ten populations of A. aphidimyza were released separately in transparent cages and their sex ratio variations were recorded every 12 hours. The primary sex ratio in this species seems to be slightly male‐biased (52.41% males), however early emergence of males biases the sex ratio up to 72% males in a few hours after emergence. Shortly after the emergence of females, the sex ratio reaches its primary situation, but as a result of male‐biased mortality after mating, the proportion of females increases gradually to 97% by the fourth and fifth days after emergence. These results explicitly suggest that direct estimation of sex ratio in natural populations may be affected by some secondary factors such as differential mortality of sexes, protandry, and differential distribution of males and females over time and/or across habitat.     

Association mapping and meta-analysis: two complementary approaches for the detection of reliable Septoria tritici blotch quantitative resistance in bread wheat (Triticum aestivum L.)

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola, is one of the most ubiquitous and important diseases of bread wheat worldwide. The aim of this study was to identify markers linked to loci conferring resistance to STB from seven biparental populations. Linkage analysis, meta-analysis and association mapping were combined to identify robust quantitative trait loci (QTLs) for resistance. Linkage analysis led to the detection of 115 QTLs for resistance to STB and 66 QTLs linked to plant height and/or earliness. Meta-analysis clustered these 115 QTLs into 27 Meta-QTLs (MQTLs) of pathogen resistance, of which 14 were found to be linked to plant height and/or earliness. Both the relationship between dwarfing and susceptibility to STB and the significant negative correlation between earliness and STB symptoms were confirmed. Eleven loci were linked to STB resistance by association mapping using a general linear model and/or a mixed linear model, of which eight co-located with STB MQTLs and two co-located with individual QTLs. Associated markers located in MQTL regions enhanced the relevance of the results and validated the potential of an association mapping approach. With several biparental populations, meta-analysis is the most relevant form of genetic analysis study, but association mapping can be used as a validation method. Regions linked to resistance in both methods should be relevant for use in breeding programs for improving resistance to STB in wheat varieties. The main interest in comparing both approaches is to detect robust loci that will be functional in many genetic backgrounds rather than just in one or a few specific backgrounds.     

Thermodynamic of Water Sorption of Grape Seed: Temperature Effect of Sorption Isotherms and Thermodynamic Characteristics

After determination of sorption isotherms of grape seeds using gravimetric method, five models with temperature effect were used to fit water sorption isotherms of grape seeds to investigate temperature effect on sorption isotherms and its thermodynamic characteristics. Halsey model had minimum mean relative percentage error (M _e ) and all other models used were good in fitting experimental data (with M _e of less than 10 %). Differential parameters such as net isosteric heat, isosteric heat, differential entropy and integral function such as equilibrium heat, net equilibrium heat, integral entropy and surface potential have been calculated. The net isosteric heat, isosteric heat and differential entropy decreased with moisture content. The net equilibrium enthalpy, equilibrium enthalpy and integral entropy decreased with moisture content. The surface potential at four temperatures (35, 45, 55 and 65 °C) was estimated, and low temperature effect was reported.     

Plasmonically Induced Energy Flow in Monodisperse Quantum Dot Solids

We studied the excitation-power-dependent red shift and broadening of the emission spectra of monodisperse CdSe/ZnS quantum dot solids when they are in close proximity of gold metallic nanoparticles. Our results suggest that these features are the signs of plasmonic enhancement of the interdot energy transfer in such solids leading to (a) efficient funneling of excitons to the locations where quantum dots with large CdSe cores are and (b) near complete depletion of excitons in regions where the quantum dots with incomplete shells or/and small core sizes are located. We studied the impacts of the heat generated by the metallic nanoparticles and discussed the effects of excitation-power-dependent photoionization rates. The uneven spatial distribution of excitons in monodisperse quantum dot solids in the presence of metallic nanoparticles suggests that plasmonic fields can drive significant spatial migration of energy in such structures.     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

λ Phage Nanobioparticle Expressing Apoptin Efficiently Suppress Human Breast Carcinoma Tumor Growth In Vivo

Using phages is a novel field of cancer therapy and phage nanobioparticles (NBPs) such as λ phage could be modified to deliver and express genetic cassettes into eukaryotic cells safely in contrast with animal viruses. Apoptin, a protein from chicken anemia virus (CAV) has the ability to specifically induce apoptosis only in carcinoma cells. We presented a safe method of breast tumor therapy via the apoptin expressing λ NBPs. Here, we constructed a λ ZAP-CMV-apoptin recombinant NBP and investigated the effectiveness of its apoptotic activity on BT-474, MDA-MB-361, SKBR-3, UACC-812 and ZR-75 cell lines that over-expressing her-2 marker. Apoptosis was evaluated via annexin-V fluorescent iso-thiocyanate/propidium iodide staining, flow-cytometric method and TUNEL assay. Transfection with NBPs carrying λ ZAP-CMV-apoptin significantly inhibited growth of all the breast carcinoma cell lines in vitro. Also nude mice model implanted BT-474 human breast tumor was successfully responded to the systemic and local injection of untargeted recombinant λ NBPs. The results presented here reveal important features of recombinant λ nanobioparticles to serve as safe delivery and expression platform for human cancer therapy.