Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation

Development of inexpensive and simple culture media is always favorable for recombinant protein over-expression in E. coli. The effects of medium composition on the production of recombinant human granulocyte-colony stimulating factor (rh-GCSF) were investigated in batch culture of E. coli BL21 (DE3) [pET23a-hgcsf]. First, the optimum medium for production of rh-GCSF was determined; and, then it was shown that mixture of amino acid addition at induction time, which was determined on the basis of amino acids frequency in the recombinant protein, increases recombinant protein expression level significantly. Furthermore, the effect of glucose concentration on productivity of rh-GCSF was investigated; 20 g/l of glucose will result in maximum attainable biomass and rh-GCSF in this process. At optimum conditions, a cell dry weight of 10.5 g/l, an expression level of about 35% of total cellular protein, rh-GCSF concentration of 1.75 ± 0.1 g/l, and overall rh-GCSF yield of 165 ± 5 mg/g were obtained.     

Gastric xanthoma: a diagnostic problem on brushing cytology smears

OBJECTIVE: To describe the cytologic findings of gastric xanthomas and to compare them with the findings of signet-ring adenocarcinoma because atypical xanthoma cells can be easily confused with signet-ring adenocarcinoma cells. STUDY DESIGN: Five cases of gastric xanthoma that were confirmed by biopsy reports were selected for study. The patients' ages ranged between 50 and 58 years; 4 were men and 1 was a woman. Twenty-one cases of signet-ring adenocarcinoma confirmed by biopsy reports were selected for comparison. Special staining was performed. RESULTS: The brushing cytology smears of the 5 cases of xanthoma revealed atypical cells, so initially they were reported as suspicious for signet-ring adenocarcinoma and biopsy examination advised. After learning of the histologic diagnosis of xanthoma, we performed special staining. The xanthoma cells were negative with periodic acid-Schiff (PAS) stain but showed a positive reaction with Oil red 0 and weakly positive reaction with Masson trichrome. Signet-ring adenocarcinoma cells showed a strongly positive reaction with PAS stain. CONCLUSION: Gastric brushing cytology findings of xanthomas have not been described before. At times the differentiation of atypical xanthoma cells from signet-ring adenocarcinoma cells is very difficult on brushing cytology smears. In this study the nuclear changes and special stains helped differentiate the 2 lesions.     

A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK

Protein folding is directed by the sequence of sidechains along the polypeptide backbone, but despite this the developement of sidechain interactions during folding is not well understood. Here, the thiol-active reagent, dithio-nitrobenzoic acid (DTNB), is used to probe the exposure of the cysteine sidechain thiols in the kinetic folding intermediates of the N-terminal domain of phosphoglycerate kinase (N-PGK) and a number of conservative (I-, L-, or V-to-C) single cysteine variants. Rapid dilution of chemically denatured protein into folding conditions in the presence of DTNB allowed the degree of sidechain protection in any rapidly formed intermediate to be determined through the analysis of the kinetics of labelling. The protection factors derived for the intermediate(s) were generally small (<25), indicating only partial burial of the sidechains. The distribution of protection parallels the previously reported backbone amide protection for the folding intermediate of N-PGK. These observations are consistent with the hypothesis that such intermediates resemble molten globule states; i.e. with native-like backbone hydrogen bonding and overall tertiary structure, but with the sidechains that make up the hydrophobic protein core dynamic and intermittently solvent exposed. The success of the competition technique in characterizing this kinetic intermediate invites application to other model systems.     

Pancreatic β-cell regeneration: From molecular mechanisms to therapy

Pancreatic β cells are a type of cells that are present in the islets of Langerhans. These cells are highly specialized for the secretion of insulin in response to low increasing of blood glucose levels. Hence, pancreatic β cells could contribute to maintaining systemic glucose homeostasis. Increasing evidence has revealed that a variety of internal (ie, genetic and epigenetic factors) and external factors (ie, radical-oxidative stress) are involved in the protection and/or regeneration of pancreatic β cells. The pathways regulating β-cell replication have been intensely investigated. Glucose has an important role in cell cycle entry of quiescent β cells, which exerts its effect via glucose metabolism and unfolded proteins. A variety of growth factors, hormones, and signaling pathways (ie, calcium-calcineurin nuclear factor of activated T cells) are others factors that could affect β-cell replication under different conditions. Therefore, a greater understanding of the underlying pathways involved in the regeneration and protection of pancreatic β cells could lead to finding and developing new therapeutic approaches. Utilization of stem cells and various phytochemical agents have provided new aspects for preventing β-cell degeneration and stimulating the endogenous regeneration of islets. Thus, these therapeutic platforms could be used as potential therapies in the treatment of insulin-dependent diabetes mellitus. Here, we summarized the various mechanisms involved in pancreatic β-cell regeneration. Moreover, we highlighted different therapeutic approaches which could be used for the regeneration of pancreatic β cells.     

Identification of genes differentially expressed during interaction of Mexican lime tree infected with " Candidatus Phytoplasma aurantifolia"

Background  

"Candidatus Phytoplasma aurantifolia", is the causative agent of witches' broom disease in Mexican lime trees (Citrus aurantifolia L.), and is responsible for major losses of Mexican lime trees in Southern Iran and Oman. The pathogen is strictly biotrophic, and thus is completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. Therefore, we have applied a cDNA- amplified fragment length polymorphism (AFLP) approach to analyze gene expression in Mexican lime trees infected by " Ca. Phytoplasma aurantifolia".     

Diversity of aminoglycoside modifying enzyme genes among multidrug resistant Acinetobacter baumannii genotypes isolated from nosocomial infections in Tehran hospitals and their association with class 1 integrons

The aim of the present study was to investigate, for the first time, the diversity of the genes encoding aminoglycoside-modifying enzymes (AME) and their association with class 1 integrons in Iranian Acinetobacter baumannii strains. A total of 100 multidrug resistant A. baumannii, isolated from eight distinct hospitals in Tehran, were enrolled in this study. Susceptibility of these isolates to antimicrobial agents including gentamicin and amikacin was determined by E-test. Aminoglycoside resistant isolates were then tested by PCR for AME genes, including aphA6, aacC1, aacC2, aacA4, aadB, aadA1, classes 1 integron, 5'-CS-3' and typed by RAPD PCR. The rate of resistance to imipenem, meropenem, gentamicin and amikacin were 39%, 39%, 38% and 32%, respectively. Intermediate resistance phenotype to gentamicin and amikacin was observed in 2% and 5% of all the isolates, respectively. After aph6 with 90% (n = 36/40), aadA1, aacC1 and aadB with 82.5% (n = 33/40), 65% (n = 26/40) and 20% (n = 8/40) were the most prevalent AME genes among aminoglycosides resistant A. baumannii isolates. A combination of two to four different resistance genes was observed in 39 of 40 strains (97.5%), with a total of 7 different combinations. PCR of integrase genes revealed that AME gene was associated with 67% of class 1 integrons. RAPD analysis showed three predominant genotypes A (n = 20), B (n = 10) and 10 unrelated genotypes. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance in A. baumannii.     

Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells

Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFβ₁) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFβ₁ can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFβ₁ treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFβ₁ treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFβ₁-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFβ₁, in the clinical progression of NSCLC.     

Heterologous production of recombinant anti-HIV microbicide griffithsin in transgenic lettuce and tobacco lines

The present paper describes a detailed study of a highly efficient protocol to multiply the number of haploids in sugar beet production and subsequent chromosome doubling. The protocol involves an experiment investigating factorial interactions between cold pretreatment, seven genotypes of sugar beet, and kinetin to improve haploid embryo induction. In addition, the effects of color of ovules and flower bud position on haploid embryo induction were investigated. After subjecting the data to analysis of variance or Student’s t test (P?<?.05), the effect sizes of the independent variables were also estimated. Cold pretreatment was effective in stimulating the ovules. The haploid embryo induction rate for 1-week cold pretreated ovules (9.01%) was higher than that of freshly cultured ones (6.15%). In comparison with hormone-free medium (5.16%), the gynogenesis rate for the media supplemented with 0.05 or 0.5 mg L^?l kinetin increased to 7.58 and 10.05%, respectively. The genotype responses were significantly different. Interactions of kinetin?×?cold pretreatment, genotype?×?hormonal treatment, genotype?×?cold pretreatment, and the three-way interaction were statistically significant. Moreover, the main effects of flower bud position, ovule color, and comma-form ovule on gynogenic response were significant. After investigating the effect of 5 g L^?l colchicine for 3, 5, or 7 min on one genotype’s (SG2) specimens, all the haploid plantlets from the other genotypes were treated for 5 min as the best treatment. The paper discusses interactions of the factors, which may be interesting for others aiming to breed doubled haploid sugar beet or possibly other related plant species.     

Preparation of an injectable doxorubicin surface modified cellulose nanofiber gel and evaluation of its anti‐tumor and anti‐metastasis activity in melanoma

Cellulose nanofibers (Cel‐NFs) gel can be considered as a useful drug carrier because of its biocompatibility, high specific surface area, and high loading capacity of drugs. Injectable Cel‐NFs gel could deliver doxorubicin (DOX) for localized chemotherapy of melanoma and suppress melanoma cells migration because of the physical barrier property of Cel‐NFs. We prepared DOX surface modified Cel‐NFs (DOX‐Cel‐NFs) gel by the electrostatic attachment of DOX molecules on the surface of Cel‐NFs. The increase in the zeta potential of nanofibers and the changes in the FTIR spectra of DOX‐Cel‐NFs compared to Cel‐NFs proved this attachment. DOX‐Cel‐NFs showed nano‐fibrous structure with an average diameter of 22.32 ± 10.66 nm after analyzing using field emission scanning electron microscopy. The suitable injectability of DOX‐Cel‐NFs gel verified its promising application for the localized chemotherapy. DOX‐Cel‐NFs gel exhibited a sustained drug release manner. The cytotoxicity results showed that DOX‐Cel‐NFs were more cytotoxic against melanoma cancer cells than the free DOX during 48 h incubation period. Moreover, DOX‐Cel‐NFs gel can suppress the melanoma cancer cells migration efficiently. Thus our results emphasize the potential of DOX‐Cel‐NFs gel as a chemotherapeutic agent for local delivery of DOX in order to treat melanoma and prevent its metastasis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:537–545, 2018