Future Nanotechnology-Based Strategies for Improved Management of Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) is a recalcitrant pathogen, which can cause gastric disorders. During the past decades, polypharmacy-based regimens, such as triple and quadruple therapies have been widely used against H. pylori. However, polyantibiotic therapies can disturb the host gastric/gut microbiota and lead to antibiotic resistance. Thus, simpler but more effective approaches should be developed. Here, some recent advances in nanostructured drug delivery systems to treat H. pylori infection are summarized. Also, for the first time, a drug release paradigm is proposed to prevent H. pylori antibiotic resistance along with an IVIVC model in order to connect the drug release profile with a reduction in bacterial colony counts. Then, local delivery systems including mucoadhesive, mucopenetrating, and cytoadhesive nanobiomaterials are discussed in the battle against H. pylori infection. Afterward, engineered delivery platforms including polymer-coated nanoemulsions and polymer-coated nanoliposomes are poposed. These bioinspired platforms can contain an antimicrobial agent enclosed within smart multifunctional nanoformulations. These bioplatforms can prevent the development of antibiotic resistance, as well as specifically killing H. pylori with no or only slight negative effects on the host gastrointestinal microbiota. Finally, the essential checkpoints that should be passed to confirm the potential effectiveness of anti-H. pylori nanosystems are discussed.

Fabrication and multiscale modeling of polycaprolactone/amniotic membrane electrospun nanofiber scaffolds for wound healing.

BACKGROUND: Enhancing the efficiency of cell-based skin tissue engineering (TE) approaches is possible via designing electrospun scaffolds possessing natural materials like amniotic membrane (AM) with wound healing characteristics. Concentrating on this aim, we fabricated innovative polycaprolactone (PCL)/AM scaffolds through the electrospinning process. METHODS: The manufactured structures were characterized by employing scanning electron microscope (SEM), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, tensile testing, Bradford protein assay, etc. In addition, the mechanical properties of scaffolds were simulated by the multiscale modeling method. RESULTS: As a result of conducting various tests, it was concluded that the uniformity and distribution of fibers decreased with an increase in the amniotic content. Furthermore, PCL-AM scaffolds contained amniotic and PCL characteristic bands. In the case of protein release, greater content of AM led to the release of higher amounts of collagen. Tensile testing revealed that scaffolds' ultimate strength increased when the AM content augmented. The multiscale modeling demonstrated that the scaffold had elastoplastic behavior. In order to assess cellular attachment, viability, and differentiation, human adipose-derived stem cells (ASCs) were seeded on the scaffolds. In this regard, SEM and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays showed significant cellular proliferation and viability on the proposed scaffolds, and these analyses illustrated that higher cell survival and adhesion could be achieved when scaffolds possessed a larger amount of AM. After 21 days of cultivation, particular keratinocyte markers, such as keratin I and involucrin, were identified through utilizing immunofluorescence and real-time polymerase chain reaction (PCR) tests. The markers' expressions were higher in the PCL-AM scaffold with a ratio of 90:10 v v-1 compared with the PCL-epidermal growth factor (EGF) structure. Moreover, the presence of AM in the scaffolds resulted in the keratinogenic differentiation of ASCs even without employing EGF. Consequently, this state-of-the-art experiment suggests that the PCL-AM scaffold can be a promising candidate in skin bioengineering. CONCLUSION: This study showed that mixing AM with PCL, a widely used polymer, in different concentrations can overcome PCL disadvantages such as high hydrophobicity and low cellular compatibility.

Biomimetic vascular tissue engineering by decellularized scaffold and concurrent cyclic tensile and shear stresses.

Decellularization by chemical approaches has harmful effects on extracellular matrix (ECM) proteins, and damages lots of functional peptides and biomolecules present in the ultrastructure. In this study, we employed a combination of chemical and physical decellularization methods to overcome these disadvantages. The induced osmotic pressure by hypertonic/hypotonic solutions dissociated and removed most of cellular membranes significantly without any detergent or chemical agent. In total, 0.025% trypsin solution was found adequate to remove the remaining debrides, and ultimately 1% Triton X-100 was utilized for final cleansing. In addition, conducting all the decellularization processes at 4 °C yielded an ECM with least damages in the ultrastructure which could be inferred by close mechanical strength and swelling ratio to the native vessel, and high quality and quantity of cell attachment, migration and proliferation which were examined by optical microscopy and scanning electron microscopy (SEM) of the histology samples. Moreover, the obtained biological scaffold (BS) had no cytotoxicity according to the MTT assay, and this scaffold is storable at -20 °C. Employing bioreactor for concurrent cyclic tensile and shear stresses improved the cell migration into pores of the BS and made the cells and the scaffold compact in analogous to native tissue. As opening angle test showed by decellularizing of the blood vessel, the residual stress dropped significantly which revealed the role of cells in the amount of induced stress in the structure. However, intact and healthy ECM explicitly recovered upon recellularization and beat the initial residual stress of the native tissue. The tensile test of the blood vessels in longitudinal and radial directions revealed orthotropic behavior which can be explained by collagen fibers direction in the ECM. Furthermore, by the three regions of the stress-strain curve can be elucidated the roles of cells, elastin and collagen fibers in mechanical behavior of the vascular tissues.

Enhancement of keratinocyte growth factor potential in inducing adipose-derived stem cells differentiation into keratinocytes by collagen-targeting.

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.

Improving bactericidal performance of implant composite coatings by synergism between Melittin and tetracycline.

Methicillin resistance Staphylococcus aureus bacteria (MRSA) are serious hazards of bone implants. The present study was aimed to use the potential synergistic effects of Melittin and tetracycline to prevent MRSA associated bone implant infection. Chitosan/bioactive glass nanoparticles/tetracycline composite coatings were deposited on hydrothermally etched titanium substrate. Melittin was then coated on composite coatings by drop casting method. The surfaces were analyzed by FTIR, XRD, and SEM instruments. Tetracycline in coatings revealed multifunctional behaviors include bone regeneration and antibacterial activity. Releasing ALP enzyme from MC3T3 cells increased by tetracycline, so it is suitable candidate as osteoinductive and antibacterial agent in orthopedic implants coatings. Melittin increased the proliferation of MC3T3 cells. Composite coatings with combination of tetracycline and Melittin eradicate all MRSA bacteria, while coatings with one of them could no t eradicate all of the bacteria. In conclusion, chitosan/bioactive glass/tetracycline/Melittin coating can be suggested as a multifunctional bone implant coating because of its osteogenic and promising antibacterial activity. Graphical abstract.

Cabbage-derived three-dimensional cellulose scaffold-induced osteogenic differentiation of stem cells.

Herbal-derived three-dimensional scaffolds have a unique structure that represents the natural cellular microenvironment and can be potentially used for tissue engineering applications. In the present study, cabbage (Cb) leaves were decellularized and then their characteristics, such as surface roughness, wettability, porosity, mechanical properties, and specific surface area, were investigated. After that, scaffold osteoinductivity was studied by bone-marrow-derived mesenchymal stem cells (BM-MSCs) osteogenic differentiation while growing on the decellularized Cb leaves. Cells mineralization, calcium secretion, alkaline phosphatase (ALP) activity, and expression levels of bone-related genes were determined during the differentiation process. Our results from the structural characterization of the scaffolds demonstrated that decellularized Cb leaves are good candidates for bone differentiation in terms of surface roughness, mechanical properties, and interconnected pores. Osteogenic differentiation evaluation of the BM-MSCs determined that the cell's ALP activity and mineralization were increased significantly while cultured on the decellularized Cb leaves compared to the cells cultured on the culture plate as a control. Besides, Runx2, ALP, collagen-1 (Col-I), and osteocalcin genes were expressed in cells cultured on decellularized Cb leaves significantly higher than cells cultured on the culture plate. Based on these results, it can be concluded that the decellularized Cb scaffold has great potential for promoting BM-MSCs proliferation and osteogenic differentiation.

Prevention the formation of biofilm on orthopedic implants by melittin thin layer on chitosan/bioactive glass/vancomycin coatings.

Methicillin-resistant and Vancomycin-resistant Staphylococcus aureus bacteria (MRSA and VRSA, respectively) can seriously jeopardizes bone implants. This research aimed to examine the potential synergistic effects of Melittin and vancomycin in preventing MRSA and VRSA associated bone implant infections. Chitosan/bioactive glass nanoparticles/vancomycin composites were coated on hydrothermally etched titanium substrates by casting method. The composite coatings were coated by Melittin through drop casting technique. Melittin raised the proliferation of MC3T3 cells, making it an appropriate option as osteoinductive and antibacterial substance in coatings of orthopedic implants. Composite coatings having combined vancomycin and Melittin eliminated both planktonic and adherent MRSA and VRSA bacteria, whereas coatings containing one of them failed to kill the whole VRSA bacteria. Therefore, chitosan/bioactive glass/vancomycin/Melittin coating can be used as a bone implant coating because of its anti-infective properties.

CRISPR/Cas9 mediated GFP-human dentin matrix protein 1 (DMP1) promoter knock-in at the ROSA26 locus in mesenchymal stem cell for monitoring osteoblast differentiation.

BACKGROUND: Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase1 . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation. METHODS: In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays. RESULTS: We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs 2 . CONCLUSIONS: The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts.

Evaluation of alginate modification effect on cell-matrix interaction, mechanotransduction and chondrogenesis of encapsulated MSCs.

Mesenchymal stem cells (MSCs) are promising cell candidates for cartilage regeneration. Furthermore, it is important to control the cell-matrix interactions that have a direct influence on cell functions. Providing an appropriate microenvironment for cell differentiation in response to exogenous stimuli is a critical step towards the clinical utilization of MSCs. In this study, hydrogels consisted of different proportions of alginates that were modified using gelatin, collagen type I and arginine-glycine-aspartic acid (RGD) and were evaluated regarding their effects on mesenchymal stem cells. The effect of applying hydrostatic pressure on MSCs encapsulated in collagen-modified alginate with and without chondrogenic medium was evaluated 7, 14 and 21 days after culture, which is a comprehensive evaluation of chondrogenesis in 3D hydrogels with mechanical and chemical stimulants. Alcian blue, safranin O and dimethyl methylene blue (DMMB) staining showed the chondrogenic phenotype of cells seeded in the collagen- and RGD-modified alginate hydrogels with the highest intensity after 21 days of culture. The results of real-time PCR for cartilage-specific extracellular matrix genes indicated the chondrogenic differentiation of MSCs in all hydrogels. Also, the synergic effects of chemical and mechanical stimuli are indicated. The highest expression levels of the studied genes were observed in the cells embedded in collagen-modified alginate by loading after 14 days of exposure to the chondrogenic medium. The effect of using IHP on encapsulated MSCs in modified alginate with collagen type I is equal or even higher than using TGF-beta on encapsulated cells. The results of immunohistochemical assessments also confirmed the real-time PCR data.

Fabrication and Characterization of Core-Shell Electrospun Fibrous Mats Containing Medicinal Herbs for Wound Healing and Skin Tissue Engineering.

Nanofibrous structures mimicking the native extracellular matrix have attracted considerable attention for biomedical applications. The present study aims to design and produce drug-eluting core-shell fibrous scaffolds for wound healing and skin tissue engineering. Aloe vera extracts were encapsulated inside polymer fibers containing chitosan, polycaprolactone, and keratin using the co-axial electrospinning technique. Electron microscopic studies show that continuous and uniform fibers with an average diameter of 209 ± 47 nm were successfully fabricated. The fibers have a core-shell structure with a shell thickness of about 90 nm, as confirmed by transmission electron microscopy. By employing Fourier-transform infrared spectroscopy, the characteristic peaks of Aloe vera were detected, which indicate successful incorporation of this natural herb into the polymeric fibers. Tensile testing and hydrophilicity measurements indicated an ultimate strength of 5.3 MPa (elongation of 0.63%) and water contact angle of 89°. In-vitro biological assay revealed increased cellular growth and adhesion with the presence of Aloe vera without any cytotoxic effects. The prepared core-shell fibrous mats containing medical herbs have a great potential for wound healing applications.

Using chemical chaperones to increase recombinant human erythropoietin secretion in CHO cell line.

In recombinant protein production, over-expressed genes induce unfolded protein response (UPR), overloaded protein aggregation in endoplasmic reticulum and its expansion. In this study, we have used 16 chemicals to improve erythropoietin production in engineered CHO cells and tried to study the mechanism of reducing protein aggregation in each treatment. Endoplasmic reticulum expansion was studied through endoplasmic reticulum specific labeling with utilizing fluorescent glibenclamide and its molecular chaperones expression were studied by real-time polymerase chain reaction. The increase in the mRNA level of EPO and endoplasmic reticulum chaperones GRP78/BiP, XBP1, ATF6, and ATF4 in different chemical treatments were not related to ER expansion. On the other hand, ER expansion in beta alanine, beta cyclodextrin and taurine treatments resulted in increased EPO secretion. Dramatically increase in EPO expression in conjugated linoleic acid, spermidine, trehalose, and maltose (19, 20, 16, and 19-fold, respectively) did not increase erythropoietin productivity, but betaine which did not caused ER expansion, with minor increase in EPO gene expression increase EPO productivity. The results indicated that betaine increase EPO secretion in engineered CHO cell line without relation to ER expansion and molecular chaperones expression.

Generation and characterization of an anti-delta like ligand-4 Nanobody to induce non-productive angiogenesis.

Antibody-based targeting of angiogenesis is a key approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. It forecasts the prognosis of human malignancies and blocking its signaling can help to inhibit neovascularization and tumor metastasis. Nanobodies are the smallest antigen-binding domains of heavy chain antibodies in camelidae. The aim of this study was to develop a Nanobody against DLL4 and apply binding and functional approaches to target it. In this work, a Nanobody library against human recombinant DLL4 was developed. After panning, the periplasmic-extract (PE) of individual colonies were screened through ELISA. The interactions between Nanobody and DLL4 were assessed using immunohistochemistry and FACS. The functional assessment was carried out via tube formation assay. We selected a Nanobody (3Nb3) with a high binding signal to DLL4, associated with a binding affinity of 3.6 nM. It was demonstrated that 3Nb3 binds to native DLL4 on the surface of MKN cells and gastric carcinoma tissue, and also inhibits the maturation of capillary-like structures in HUVECs. The results were indicative of the potential of Nanobody for DLL4 identification and can broaden the scope for development of cancer diagnosis and treatment techniques.

Development and characterization of a camelid single-domain antibody directed to human CD22 biomarker.

CD22 is a B-cell-specific trans-membrane glycoprotein, which is found on the surface of the most B cells and modulates their function, survival, and apoptosis. Recently, targeting this cell surface biomarker in B-cell malignancies and disorders has attracted a lot of attention. The variable domain of camelid single-chain antibodies (VHH, nanobody) is a form of antibodies with novel properties including small size (15-17 kDa), thermal and chemical stability, high affinity and homology to human antibody sequences. In this study, a novel anti-CD22-specific VHH (Nb) has been developed and characterized by the screening of an immunized phage display library and its binding to CD22+ B cells is evaluated. Produced anti-CD22 VHH had a single protein band about 17 kDa of molecular size in Western blotting and its binding affinity was approximately 9 × 10-9  M. Also, this product had high specificity and it was able to recognize the natural CD22 antigen in CD22+ cell lysate as well as on the cell surface (93%). This anti-CD22 VHH with both high affinity and specificity recognizes CD22 antigen well and can be used in diagnosis and treatment of B cell disorders and malignancies.

Mechanical and Chemical Predifferentiation of Mesenchymal Stem Cells Into Cardiomyocytes and Their Effectiveness on Acute Myocardial Infarction.

Myocardial infarction is one of the leading causes of death all over the world. Mesenchymal stem cells (MSCs) transplantation has shown a promising potential to recovery of ischemic heart disease due to their capability in differentiating into cardiac cells. However, various investigations have been performed to optimize the efficacy of cardiac cell therapy in recent years. Here, we sought to interrogate the effect of autologous transplantation of undifferentiated and predifferentiated adipose and bone marrow-derived MSCs in a rabbit model of myocardial infarction and also to investigate whether cardiac function could be improved by mechanically induced MSCs via equiaxial cyclic strain. The two sources of MSCs were induced toward cardiomyocyte phenotype using mechanical loading and chemical factors and thereafter injected into the infarcted myocardium of 35 rabbits. Echocardiography and histopathology studies were used to evaluate cardiac function after 2 months. The results demonstrated significant scar size reduction and greater recovery of left ventricle ejection fraction after transplantation of predifferentiated cells, though the differences were not significant when comparing mechanically with chemically predifferentiated MSCs. Thus, although there was no significant improvement in infarcted myocardium between chemically and mechanically predifferentiated MSCs, mechanically induced cells are more preferred due to lack of any chemical intervention and cost reasonableness in their preparation method. Outcomes of this study may be useful for developing future therapeutic strategies, however long-term assessments are still required to further examine their effectiveness.

Sustained release of TGF-ß1 via genetically-modified cells induces the chondrogenic differentiation of mesenchymal stem cells encapsulated in alginate sulfate hydrogels.

Strategies based on growth factor (GF) delivery have attracted considerable attention in tissue engineering applications. Among different GFs, transforming growth factor beta 1 (TGF-ß1) is considered to be a potent factor for inducing chondrogenesis. In the present study, an expression cassette encoding the TGF-ß1 protein was prepared and transfected into the SP2/0-Ag14 cell line. The confocal microscopy of the transfected cells was performed to confirm the correct transfection process. The expression and in vitro release kinetics of the recombinant TGF-ß1 were assessed by western blot analysis and ELISA, respectively. Moreover, the biological activity of the expressed protein was compared with that of a commercially available product. The chondrogenic effects of the sustained release of the recombinant TGF-ß1 in an in vitro co-culture system were evaluated using a migration assay and real-time PCR. Results of confocal microscopy confirmed the successful transfection of the vector-encoding TGF-ß1 protein into the SP2/0-Ag14 cells. The bioactivity of the produced protein was in the range of the commercial product. The sustained release of the TGF-ß1 protein via SP2/0-Ag14 cells encapsulated in hydrogels encouraged the migration of adipose-derived MSCs. In addition, the expression analysis of chondrogenesis-related genes revealed that the pretreatment of encapsulated Ad-MSCs cells in alginate sulfate hydrogels through their exposure to the sustained release of TGF-ß1 is an efficient approach before transplantation of cells into the body.

An antibody fragment against human delta-like ligand-4 for inhibition of cell proliferation and neovascularization.

OBJECTIVES: Angiogenesis targeting is an attractive approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in neovascular development and its inhibitors have recently entered clinical trials for solid tumors. The aim of this study was to evaluate the possibilities of using anti-DLL4 antibody fragment as an angiogenesis maturation inhibitor. MATERIALS AND METHODS: In this study, a DLL4-specific Nanobody, named 3Nb3, was selected and assessed by western blotting and internalization assays. Functional assessments included MTT, apoptosis, and chicken chorioallantoic membrane (CAM) assays. RESULTS: Based on the results, 3Nb3 specifically binds to DLL4 and internalizes into MKN cell. Furthermore, 3Nb3 significantly inhibited the proliferation of cells and also neovascularization in the CAM. CONCLUSIONS: These data demonstrated the potential of Nanobody for application in targeting DLL4. Our findings may provide a basis for the development of novel therapeutic techniques to inhibit growth and neovascularization of tumors.

Physicochemical screening for chemical stabilizer of erythropoietin to prevent its aggregation.

Recombinant protein aggregation is a problematic issue and can provoke immunological response. The aim of this study was to analyze the stability of erythropoietin (EPO), as a therapeutic protein expressed in mammalian cells, in the presence of different chemicals and find a specific stabilizer for EPO. The effects of several chemicals, including mannitol, betaine, trehalose, taurine, linoleic acid, beta-cyclodextrin, copper sulfate, spermidine, maltose, maltodextrin, sucrose, dextran, beta-alanine, myo-inositol, and cysteine, on protein stabilization through the thermally induced aggregation of EPO were monitored. Based on the results of turbidity assay for thermal aggregation, three different patterns were observed for protein stability of active pharmaceutical ingredient of EPO, namely, accelerated, dose-dependent, and inhibitory behaviors for aggregate formation due to treatment with spermidine, mannitol, and betaine, respectively. According to circular dichroism outcomes, EPO treatment with betaine and spermidine resulted in different helical contents of the secondary structure. Dynamic light scattering experiments indicated that treating EPO with betaine resulted in less protein aggregation due to freeze and thaw stresses. Betaine was able to stabilize EPO and inhibit its aggregation, as opposed to spermidine that induced protein aggregation.

Proteomic analysis of drug-resistant Mycobacterium tuberculosis by one-dimensional gel electrophoresis and charge chromatography.

Multidrug-resistant tuberculosis (MDR-TB) is a form of TB caused by Mycobacterium tuberculosis (M. tuberculosis) that do not respond to, at least, isoniazid and rifampicin, the two most powerful, first-line (or standard) anti-TB drugs. Novel intervention strategies for eliminating this disease were based on finding proteins that can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profiles of MDR-TB with sensitive isolates. Proteomic analysis of M. tuberculosis MDR-TB and sensitive isolates was obtained with ion exchange chromatography coupled with MALDI-TOF-TOF (matrix-assisted laser desorption/ionization) in order to identify individual proteins that have different expression in MDR-TB to be used as a drug target or diagnostic marker for designing valuable TB vaccines or TB rapid tests. We identified eight proteins in MDR-TB isolates, and analyses showed that these proteins are absent in M. tuberculosis-sensitive isolates: (Rv2140c, Rv0009, Rv1932, Rv0251c, Rv2558, Rv1284, Rv3699 and MMP major membrane proteins). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug-resistant and sensitive M. tuberculosis isolates.

Fish-Oil-Derived DHA-mediated Enhancement of Apoptosis in Acute Lymphoblastic Leukemia Cells is Associated with Accumulation of p53, Downregulation of Survivin, and Caspase-3 Activation.

In acute lymphoblastic leukemia (ALL), resistance to chemotherapy is associated with inactivation of p53 and upregulation of survivin. Thus, targeting the p53 and survivin expression may provide an attractive strategy for ALL treatment. It has been shown that fish-oil-derived docosahexaenoic acid (DHA) activates several antitumorigenic mechanisms in tumor cells, but little is known regarding the role of DHA on modulating p53 and survivin expression in ALL cells. In this study, we investigated the alterations of the p53 and survivin expression and induction of apoptosis in DHA-treated Molt-4 cells that serve as a model for ALL cells. Molt-4 cells were treated with 50, 100, 150, and 200 µM DHA after which cell proliferation, survivin mRNA and protein levels, p53 protein level, caspase-3 activation, and apoptotic rates were evaluated by different cellular and molecular techniques. After 48- and 72-h treatments with DHA at concentrations ranging from 50 to 200 µM, cell proliferation rates were measured to be 80.5-44.4%, and 73.4-14.4%, respectively, compared to untreated cells. We also found that treatment for 48 h with 200 µM DHA resulted in 10.8- and 3.6-fold increase in p53 protein level and caspase-3 activation followed by 4.7-and 1.6-fold decrease in survivin mRNA and protein levels, respectively, compared to untreated cells. Treatment of cells with different concentrations of DHA dramatically increased the p53/survivin and caspase-3/survivin ratios by 2.8- to 16.9-fold and 3.3 to 5.6-fold increases, respectively, compared to untreated cells. A decrease in the number of cells ranging from 16% to 70% and an increase in the number of apoptotic cells ranging from 9.3% to 93% was also observed with increasing DHA concentrations. In conclusion, p53 and survivin may provide promising targets of DHA in ALL cells and this compound with high proapoptotic capacity represents the possibility of its therapeutic application for ALL treatment.

Flow Preconditioning of Endothelial Cells on Collagen-Immobilized Silicone Fibers Enhances Cell Retention and Antithrombotic Function.

Stability and antithrombotic functionality of endothelial cells on silicone hollow fibers (SiHFs) are critical in the development of biohybrid artificial lungs. Here we aimed to enhance endothelial cell retention and anti-thrombotic function by low (12 dyn/cm2 , 24 h) fluid shear stress ("flow") preconditioning of endothelial cells seeded on collagen-immobilized SiHFs. The response of endothelial cells without preconditioning (48 h static culture) and with preconditioning (24 h static culture followed by 24 h flow preconditioning) on hollow fibers to high fluid shear stress (30 dyn/cm2 , 1 h) was assessed in a parallel-plate flow chamber. Finite element (FE) modeling was used to simulate shear stress within the flow chamber. We found that collagen immobilization on hollow fibers using carbodiimide bonds provided sufficient stability to high shear stress. Flow preconditioning for 24 h before treatment with high shear stress for 1 h on collagen-immobilized hollow fibers increased cell retention (1.3-fold). The FE model showed that cell flattening due to flow preconditioning reduced maximum shear stress on cells by 32%. Flow preconditioning prior to exposure to high fluid shear stress enhanced the production of nitric oxide (1.3-fold) and prostaglandin I2 (1.2-fold). In conclusion, flow preconditioning of endothelial cells on collagen-immobilized SiHFs enhanced cell retention and antithrombotic function, which could significantly improve current biohybrid artificial lungs.