Optimal Cocktail Research Articles

Synergistic mechanism of GH11 xylanases with different action modes from Aspergillus niger An76

BackgroundXylan is the most abundant hemicellulose polysaccharide in nature, which can be converted into high value-added products. However, its recalcitrance to breakdown requires the synergistic action of multiple enzymes. Aspergillus niger, possessing numerous xylan degrading isozyme-encoding genes, are highly effective xylan degraders in xylan-rich habitats. Therefore, it is necessary to explore gene transcription, the mode of action and cooperation mechanism of different xylanase isozymes to further understand the efficient xylan-degradation by A. niger.ResultsAspergillus niger An76 encoded a comprehensive set of xylan-degrading enzymes, including five endo-xylanases (one GH10 and four GH11). Quantitative transcriptional analysis showed that three xylanase genes (xynA, xynB and xynC) were up-regulated by xylan substrates, and the order and amount of enzyme secretion differed. Specifically, GH11 xylanases XynA and XynB were initially secreted successively, followed by GH10 xylanase XynC. Biochemical analyses displayed that three GH11 xylanases (XynA, XynB and XynD) showed differences in catalytic performance and product profiles, possibly because of intricate hydrogen bonding between substrates and functional residues in the active site architectures impacted their binding capacity. Among these, XynB had the best performance in the degradation of xylan and XynE had no catalytic activity. Furthermore, XynA and XynB showed synergistic effects during xylan degradation.ConclusionsThe sequential secretion and different action modes of GH11 xylanases were essential for the efficient xylan degradation by A. niger An76. The elucidation of the degradation mechanisms of these xylanase isozymes further improved our understanding of GH-encoding genes amplification in filamentous fungi and may guide the design of the optimal enzyme cocktails in industrial applications.

Conditioned medium produced by fibroblasts cultured in low oxygen pressure allows the formation of highly structured capillary-like networks in fibrin gels

Tissue engineering is an emerging and promising concept to replace or cure failing organs, but its clinical translation currently encounters issues due to the inability to quickly produce inexpensive thick tissues, which are necessary for many applications. To circumvent this problem, we postulate that cells secrete the optimal cocktail required to promote angiogenesis when they are placed in physiological conditions where their oxygen supply is reduced. Thus, dermal fibroblasts were cultivated under hypoxia (2% O2) to condition their cell culture medium. The potential of this conditioned medium was tested for human umbilical vein endothelial cell proliferation and for their ability to form capillary-like networks into fibrin gels. The medium conditioned by dermal fibroblasts under hypoxic conditions (DF-Hx) induced a more significant proliferation of endothelial cells compared to medium conditioned by dermal fibroblasts under normoxic conditions (DF-Nx). In essence, doubling time for endothelial cells in DF-Hx was reduced by 10.4% compared to DF-Nx after 1 week of conditioning, and by 20.3% after 2 weeks. The DF-Hx allowed the formation of more extended and more structured capillary-like networks than DF-Nx or commercially available medium, paving the way to further refinements.

Reversible TET Inhibitor As a Hematopoietic Stem Cell Booster: A Novel Strategy to Improve Stem Cell Function

Hematopoietic stem cells (HSCs) are responsible for the adaptation capacity in times of need but are also subjected to disease processes, natural or iatrogenic damage, and age-related attrition. The latter can lead to deficient production, decreased compensatory capacity and degenerative diseases such as MDS. In childhood, hereditary BMF predominates but increasingly with age, acquired idiopathic AA is a leading cause of HSC failure. Throughout life iatrogenic cases and cumulative exposures to environmental toxicities may lead to failure of the HSC compartment. Pharmacologic HSC boosters capable of expanding HSCs would have a wide range of clinical applications in acquired and inherited BMF states, including reconstitution of exhausted hematopoiesis after chemotherapy, aging, or HSC grafting. Currently, hematopoietic growth factors (HGF) are used but lead to progenitor rather than HSC expansion despite their success in clinical application. Depletion of TET2 in murine models leads to impairment of cellular differentiation and increases the proportion of HSCs and progenitors suggesting that TET2 is a key regulator of hematopoietic homeostasis and HSC self-renewal. Alterations of TET2 via somatic mutations and/ or deletion are frequent in MDS whereby HSC and progenitor expansion may be a key component of neoplastic evolution. We hypothesized that chemical agents reversibly inhibiting TET2 activity might phenocopy HSC expansion due to mutations and be applied as HSC boosters. Using structure guided approaches, we designed, synthesized and subsequently optimized bioavailable TET inhibitors (TETi). Among them, the one of the most effective TETi, named TETi76, showed dose-dependent inhibitory activity against TET dioxygenases in in vitro cell-free and cell culture systems with 5hmC production as a read out. We developed esterified forms of TETi76 and used in our cellular experimental models. Esterified TETi is bioavailable and non-toxic to normal bone marrow (BM) cells in therapeutically effective doses. Treatment of TETi76 resulted in a 44+15% and 42+17% increase in the clonogenic potential of human and murine BM cells consistent with the proliferative advantage gained by loss of TET activity in HSCs. We then performed serial replating experiments to determine the effect of TET inhibition on immature hematopoietic progenitor cells. Over 3 consecutive passages, TETi76 treatment prolonged the durability and capacity of human HSCs to maintain colony-forming cells (155+24 vs.107+14 colonies per 1x105 P1 cells). As long-term culture initiating cells (LTC-IC) are the best in vitro surrogates of HSCs, we also investigated the effect of TETi76 in LTC-IC cultures (n=3). The weekly addition of 1μM-TETi76 resulted in nearly 2-fold increase in LTC-IC numbers at the end of the culture (116±27 vs. 64 ±26 colonies per 2x106 P1 cells, p=.011). Expansion of grafts e.g., in the setting of umbilical cord HSC transplant (UCHSC) could be an important medical area of application of TETi76. We performed suspension cultures (n=3) with an optimal cocktail of hematopoietic growth factors (HGF) in the presence or absence of TETi76 (1μM). In control cultures, total cellular output peaked on day 14, but in the presence of TETi, growth continued beyond day 28. Cumulatively, total cellular output per 106 input was 27+2.61 x106cells/mL in control cultures and 32+0.642 x106 cells/mL in TETi treated cultures on day 28. CD34+ cell output was significantly higher in cultures treated with TETi vs. vehicle (2.5x105vs. 0.9X106)CD34+ cells per 1x104 CD34+ cell input). Similar effects were observed in murine BM suspension cultures. BM cells from C57BL/6 mice (n=3) were supplemented with HGF± TETi76 (1μM). TETi treatment led to a 5-fold HSC expansion compared to vehicle treated cells at 20 days of culture. The effect of TETi76 was reversed by treatment with ascorbic acid (50 μM), a known TET activator. Cumulatively our in vitro results suggest that the presence of TETi prevents exhaustion of immature cells, observed with growth factor driven expansion. In summary, our study indicates that novel agents modulating TET activity prevent exhaustion of HSC and may help expand the HSC in vitro. In vivo experiments examining the effects of TETi on hematopoietic recovery following radiation-induced aplasia, and competitive transplant experiments of grafts exposed in vivo and in vitro to TETi are underway. Disclosures Sekeres: Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Production of lactic acid from industrial waste paper sludge using Rhizopus oryzae MTCC5384 by simultaneous saccharification and fermentation

Petrochemical plastics have become a basic requisite in today’s world. Their production has increased dramatically since the last few decades owing to the increase in their demand. Sustainable green plastics produced from polylactic acid have proven to be an alternative to conventional plastics pertaining to their tendency of biodegradability; they are disintegrated by microorganisms and are utilized as carbon sources by plants and other primitive organisms. Rhizopus species is appropriate for the biosynthesis of lactic acid, which is the basic raw material of polylactic acid could be obtained from lignocellulosic biomass since the fungus has the ability to grow on and ferment various carbon sources like cellulose, starch and so on. This research paper deals with the effective biosynthesis of lactic acid from Paper Sludge (PS) through the method of simultaneous saccharification and fermentation (SSF), along with the optimization of various parameters like temperature, pH and initial substrate concentration. An economical and an alternative methodology was opted to prepare cellulase enzyme necessary for the fermentation process, considering the fact that commercially available cellulase are exorbitant. Various combinations of the above enzymes were tested to determine the optimal cellulase cocktail suitable for these conditions. Finally, lactic acid productivity of 27 g/L was achieved when using a blended cellulase cocktail at 50:50 proportion of commercial and fabricate cellulase using a feed concentration of 75 g/L at 40 °C and 6 pH after144 hr by SSF.

Choice models with mixtures: An application to a cocktail experiment

Data from choice experiments are analyzed using choice models. Proper modeling allows a good understanding of the consumer preferences, the identification of consumer segments and the optimization of products, and, eventually, offers opportunities for competitive advantage. However, in the analysis of choice data, products that are mixtures of ingredients have been largely overlooked. In this paper, we combine traditional choice models and traditional mixture models. We apply the resulting mixture choice models to data from a real-life experiment in which consumers made pair-wise comparisons involving seven cocktails. For the choice models, we first assume consumer homogeneity. Next, we allow for heterogeneity among individuals. Therefore, we discuss the multinomial logit model, the mixed logit model and the latent class model. For identifying segments, besides the latent class model, we explore various two-stage segmentation approaches in which forces, hierarchical Bayes estimates and Firth individual-level estimates are used as input for a cluster analysis. The results show that the mixed logit model and the latent class model describe the data better than the multinomial logit model. The optimal cocktails for the segments we identified using the various methods are not identical, but show many similarities.

Screening of probiotics suitable for fermentation of grains

Data from choice experiments are analyzed using choice models. Proper modeling allows a good understanding of the consumer preferences, the identification of consumer segments and the optimization of products, and, eventually, offers opportunities for competitive advantage. However, in the analysis of choice data, products that are mixtures of ingredients have been largely overlooked. In this paper, we combine traditional choice models and traditional mixture models. We apply the resulting mixture choice models to data from a real-life experiment in which consumers made pair-wise comparisons involving seven cocktails. For the choice models, we first assume consumer homogeneity. Next, we allow for heterogeneity among individuals. Therefore, we discuss the multinomial logit model, the mixed logit model and the latent class model. For identifying segments, besides the latent class model, we explore various two-stage segmentation approaches in which forces, hierarchical Bayes estimates and Firth individual-level estimates are used as input for a cluster analysis. The results show that the mixed logit model and the latent class model describe the data better than the multinomial logit model. The optimal cocktails for the segments we identified using the various methods are not identical, but show many similarities.

Perfused 3D angiogenic sprouting in a high-throughput in vitro platform

Angiogenic sprouting, the growth of new blood vessels from pre-existing vessels, is orchestrated by cues from within the cellular microenvironment, such as biochemical gradients and perfusion. However, many of these cues are missing in current in vitro models of angiogenic sprouting. We here describe an in vitro platform that integrates both perfusion and the generation of stable biomolecular gradients and demonstrate its potential to study more physiologically relevant angiogenic sprouting and microvascular stabilization. The platform consists of an array of 40 individually addressable microfluidic units that enable the culture of perfused microvessels against a three-dimensional collagen-1 matrix. Upon the introduction of a gradient of pro-angiogenic factors, the endothelial cells differentiated into tip cells that invaded the matrix. Continuous exposure resulted in continuous migration and the formation of lumen by stalk cells. A combination of vascular endothelial growth factor-165 (VEGF-165), phorbol 12-myristate 13-acetate (PMA), and sphingosine-1-phosphate (S1P) was the most optimal cocktail to trigger robust, directional angiogenesis with S1P being crucial for guidance and repetitive sprout formation. Prolonged exposure forces the angiogenic sprouts to anastomose through the collagen to the other channel. This resulted in remodeling of the angiogenic sprouts within the collagen: angiogenic sprouts that anastomosed with the other perfusion channel remained stable, while those who did not retracted and degraded. Furthermore, perfusion with 150 kDa FITC-Dextran revealed that while the angiogenic sprouts were initially leaky, once they fully crossed the collagen lane they became leak tight. This demonstrates that once anastomosis occurred, the sprouts matured and suggests that perfusion can act as an important survival and stabilization factor for the angiogenic microvessels. The robustness of this platform in combination with the possibility to include a more physiological relevant three-dimensional microenvironment makes our platform uniquely suited to study angiogenesis in vitro.

Premedication for Endotracheal Intubation in the Neonate.

Endotracheal intubation, a common procedure in neonatal intensive care, results in distress and disturbs physiologic homeostasis in the newborn. Analgesics, sedatives, vagolytics, and/or muscle relaxants have the potential to blunt these adverse effects, reduce the duration of the procedure, and minimize the number of attempts necessary to intubate the neonate. The medical care team must understand efficacy, safety, and pharmacokinetic data for individual medications to select the optimal cocktail for each clinical situation. Although many units utilize morphine for analgesia, remifentanil has a superior pharmacokinetic profile and efficacy data. Because of hypotensive effects in preterm neonates, sedation with midazolam should be restricted to near-term and term neonates. A vagolytic, generally atropine, blunts bradycardia induced by vagal stimulation. A muscle relaxant improves procedural success when utilized by experienced practitioners; succinylcholine has an optimal pharmacokinetic profile, but potentially concerning adverse effects; rocuronium may be the agent of choice based on more robust safety data despite a relatively prolonged duration of action. In the absence of an absolute contraindication, neonates should receive analgesia with consideration of sedation, a vagolytic, and a muscle relaxant before endotracheal intubation. Neonatal units must develop protocols for premedication and optimize logistics to ensure safe and timely administration of appropriate agents.

Bioethanol production from rice straw by simultaneous saccharification and fermentation with statistical optimized cellulase cocktail and fermenting fungus

BackgroundRice straw is one of the abundant lignocellulosic biomass with potential as a feedstock for bioethanol production. To produce ethanol from the biomass biologically, enzymatic hydrolysis is necessary that can effectively degrade rice straw into fermentable sugars such as glucose and xylose. Many researches utilized many kinds of commercial cellulase reagents on the hydrolysis of cellulose. Since these have different enzyme activities, enzyme reagents suitable for each biomass must be selected. In this study, three appropriate cellulase reagents were selected by multivariate analysis technique and then optimized by design of experiments method for efficient hydrolysis of alkali-pretreated rice straw. Moreover, an ethanol production from the treated straw was performed by simultaneous saccharification and fermentation (SSF) with the optimized enzyme cocktail and xylose-fermenting fungus of Mucor circinelloides.ResultsPretreatment by alkali solution of rice straw resulted in the increase of fermentable sugar content from 56.3 to 80.0%. The desirable commercial enzyme reagents for saccharification of the straw were determined as a combination of “Cellulase Onozuka 3S”, “Cellulase T Amano 4”, and “Pectinase G Amano” by a multivariate analysis based on various cellulolytic activities of each reagent. The optimum mixing ratio was calculated by response surface method based on design of experiment method. The optimized cocktail successfully achieved 75.3 g/L in production of the total fermentable sugar by hydrolysis of alkali-treated rice straw that agreed with the hydrolysis efficiency of 94.1%. SSF of 100 g/L treated rice straw with the optimal cocktail and M. circinelloides under aerobic condition resulted in 30.5 g/L ethanol concentration for 36 h.ConclusionThe construction of cellulase cocktail by the proposed statistical method enabled efficient hydrolysis of alkali-treated rice straw. SSF process combining the optimized cocktail and a xylose-fermenting fungus could be expected as a promising system for ethanol production from various lignocellulosic biomasses.

Development of enzyme cocktails for complete saccharification of chitin using mono-component enzymes from Serratia marcescens

Abstract One potential strategy for biorefining of chitin-rich biomass entails enzymatic saccharification, which, so far, has been scarcely explored. Here, saccharification of chitin was explored using response surface methodology available in the MODDE ® 10 software, to develop optimal cocktails of five mono-component enzymes from Serratia marcescens , three chitinases, Sm ChiA, Sm ChiB, Sm ChiC, a lytic polysaccharide monooxygenase, Sm LPMO10A (or “CBP21”), and a beta- N -acetylhexosaminidase, Sm CHB (“chitobiase”). These five enzymes were recombinantly produced in Escherichia coli . For both shrimp and crab chitins, Sm ChiA was the most abundant (40% and 38%, respectively) in the optimized cocktails, whereas Sm ChiB, Sm ChiC and Sm LPMO10A were present at 30% and 26%, 15% and 23%, and 3% and 2%, respectively. Saccharification yields were 70%–75%, whereas a “minimal” cocktail of Sm ChiA and Sm CHB gave only 40% saccharification. These results show that enzymatic saccharification of chitin requires multiple enzyme activities applied at dosages similar to those used for saccharification of cellulose.

High-Order Drug Combinations Are Required to Effectively Kill Colorectal Cancer Cells

Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950-63. ©2016 AACR.

Prediction of multidimensional drug dose responses based on measurements of drug pairs

Finding potent multidrug combinations against cancer and infections is a pressing therapeutic challenge; however, screening all combinations is difficult because the number of experiments grows exponentially with the number of drugs and doses. To address this, we present a mathematical model that predicts the effects of three or more antibiotics or anticancer drugs at all doses based only on measurements of drug pairs at a few doses, without need for mechanistic information. The model provides accurate predictions on available data for antibiotic combinations, and on experiments presented here on the response matrix of three cancer drugs at eight doses per drug. This approach offers a way to search for effective multidrug combinations using a small number of experiments.

T cell receptor knockout efficiency utilizing an engineered meganuclease is influenced by stimulation conditions.

Abstract Recent reports describing allogeneic chimeric antigen receptor (CAR) T cell therapies have generated tremendous excitement over the possibility of a universal CAR T platform. We are utilizing a highly engineered meganuclease designed to target the T cell receptor (TCR) alpha chain to eliminate expression of the TCR, preventing graft versus host disease following adoptive transfer. Meganuclease cleavage efficiency is highly dependent on stimulation of T cells prior to delivery of the meganuclease. To determine optimal activation conditions for TCR knockout and cell yield, T cells were activated with Dynabeads® Human T cell Activator CD3/CD28, Immunocult™ CD3/CD28 and CD3/CD28/CD2 T Cell Activators, and MACS® GMP TransAct CD3/CD28 reagent prior to nucleofection. T cells were stained prior to and following nucleofection to identify cell activation markers and cytokine receptors (CD69, IL2R, IL7R, IL15R, IL21R). Knockout efficiency was determined by staining for CD3 expression. Our results indicate that Dynabead-stimulated T cells expressed the highest levels of activation markers at 18 hours post-stimulation, and that Dynabeads® and Immunocult™ CD3/CD28/CD2 reagents both promote robust T cell proliferation and support the highest TCR knockout efficiency following our protocol. Furthermore, expression of cytokine receptors varies both during stimulation and post-nucleofection, suggesting an optimal cocktail of cytokines may promote T cell proliferation. Future studies will focus on further optimization of T cell stimulation conditions that may promote TCR knockout and expansion of T cells post-nucleofection.

Optimizing an 18F-NaF and 18F-FDG cocktail for PET assessment of metastatic castration-resistant prostate cancer.

PET/computed tomography (CT) imaging with the sodium-(F)-fluoride/2-(F)-fluoro-2-deoxy-D-glucose (F-NaF/F-FDG) cocktail has been proposed for patients with osseous metastases. This work aimed to optimize the cocktail composition for patients with metastatic castration-resistant prostate cancer (mCRPC). The study was carried out on six patients with mCRPC, with a total of 26 analyzed lesions. The patients were injected with F-NaF and F-FDG at separate time points. Dynamic PET/CT imaging recorded the uptake time course for both the tracers into osseous metastases. F-NaF and F-FDG uptakes were decoupled by kinetic analysis, which enabled calculation of F-NaF and F-FDG standardized uptake values (SUVs) images. Peak, mean, and total SUVs were evaluated for both tracers and all visible lesions. The F-NaF/F-FDG cocktail was optimized under the assumption that the contribution of both tracers to image formation is equal. SUV images from PET/CT imaging with a combination of F-NaF and F-FDG were generated for cocktail compositions with an F-NaF : F-FDG ratio varying from 1 : 8 to 1 : 2. The F-NaF peak and mean SUVs were on average four to five times higher than the F-FDG peak and mean SUVs, with an interlesion coefficient of variations of 20%. The total SUV for F-NaF was on average seven times higher than that for F-FDG. When the F-NaF : F-FDG ratio changed from 1 : 8 to 1 : 2, the typical SUV on the generated PET images increased by 50%, whereas the change in the uptake visual pattern was hardly noticeable. F-NaF and F-FDG in the cocktail contribute equally to image formation when the F-NaF : F-FDG ratio is 1 : 5. Therefore, we propose this ratio as the optimal cocktail composition for mCRPC patients. We also urge to strictly control the cocktail composition during any F-NaF/F-FDG cocktail PET/CT examination.

Abstract 13675: New Treatments for Myocardial Stunning: Creating an Optimal Drug Cocktail for CPR

Introduction: Myocardial stunning following sudden cardiac arrest resuscitation increases morbidity and mortality. Previous work suggests that intra-ischemic cooling protects against stunning by enhancing Akt survival signaling, with related enhancement of glucose utilization and NAD+ recovery. New or existing drugs that mimic these effects could improve cardiac arrest outcomes. Here we test agents known to activate PI3K/Akt signaling, increase pyruvate dehydrogenase activity, and enhance NAD+ content. They were tested for their ability to improve contractile recovery in a mouse cardiomyocyte stunning model. Methods: Primary neonatal cardiomyocytes were exposed to 30 min ischemia and 90 min reperfusion followed by 10 min isoproterenol (1μM). Therapeutic hypothermia (TH) using intra-ischemic cooling (32 ± 0.5°C), the PTEN inhibitor VO-OHpic (VO, 1μM), insulin (0.01U/ml), intralipid (ILE, 0.25%), dichloracetate (DCA, 2 mM), and the NAD+ augmentation agents NAM (10 μM), and Nampt peptide (1-200 nM) were administered at reperfusion. Akt phosphorylation was detected by Western blot. ATP content was measured by a fluorescent assay kit. Results: Compared to control cells, spontaneous contractions ceased within 5 min of ischemia and upon reperfusion recovered to less than 40% of baseline contractions despite isoproterenol treatment. Comparable to TH, the use of VO, DCA, NAM, insulin or ILE all induced a rapid and strong contractile recovery with the most significant sustained effect seen with VO and ILE. Both VO and TH improved recovery of ATP after reperfusion to comparable levels. NAM resulted in a rapid recovery that was not sustained with one dose, while Nampt had no effect. The most synergistic cocktail of two-drug combinations tested was VO plus NAM. VO increased Akt phosphorylation while NAM treatment did not, suggesting that the complementary protective effect of NAM was downstream of Akt activation. Conclusions: Treatment of contractile stunning after cardiac ischemia can be achieved by drugs that mimic critical aspects of cooling protection, including Akt activation and NAD+ supplementation. Further work is needed to test drug combinations during cardiopulmonary resuscitation (CPR) that improve myocardial recovery.

Addition of CpG ODN and Poly (I:C) to a standard maturation cocktail generates monocyte-derived dendritic cells and induces a potent Th1 polarization with migratory capacity

Monocyte-derived dendritic cells (DCs) are used as immunoadjuvant cells in cancer vaccines and have made great progress. However, an optimal DCs subset is vital for this treatment effect, the current ′gold standard′ cytokine cocktail DCs have a shortcoming in their cytokines secretion, especially IL-12p70, mainly because of the existence of PGE2. Therefore, it is necessary to find an appropriate DCs-based immunotherapeutic protocol. In this study, we compared a novel ′improved′ maturation cytokine cocktail with the current ′gold standard′ maturation cytokine cocktail used for generating standard DCs. The ′improved′ maturation cytokine cocktail DCs showed a higher levels surface markers expression (CD80, CD83, CD86 and HLA-DR), the chemokine receptors CXCR4 and CCR7 and chemokine CCL19, CCL21 and CXCL21, whereas CCR5 expression was reduced. Most importantly, in contrast to ′gold standard′ DCs, which secrete little IL-12p70 and as a result induce mainly Th2 immunity, ′improved′ cytokine cocktail DCs secreted higher levels IL-12p70 and also secreted similar concentration IL-10. To removal of PGE2 from the ′improved′ DCs did increase the IL-12p70 production. In conclusion, we here present the ′improved′ DCs, as an optimal maturation cocktail protocol, can induce high migratory potential, generate immunostimulatory DCs, produce higher levels IL-12p70 with superior capacity to induce Th1 immunity, when compared with the ′gold standard′ DCs.

In-house cellulase production from AFEX™ pretreated corn stover using Trichoderma reesei RUT C-30

Cellulase represents a major cost for biochemical conversion of lignocellulosic biomass to biofuels. In the present study, in-house production of cellulases from Ammonia Fiber Expansion pretreated corn stover (AFEX-CS) using Trichoderma reesei Rut C-30 was investigated. Seed culture was conducted in a simple medium containing corn steep liquor (CSL) and AFEX-CS hydrolysate. The effects of fermentation medium components (CSL, AFEX-CS, Tween 80 and CaCO3) on cellulase production were investigated and a simple fermentation medium was developed containing 20 g L−1 AFEX-CS and 4.0 g L−1 CaCO3. A cellulase activity of 1.5 FPU per mL was produced in this medium. Addition of 0.5% (v/v) CSL in the fermentation medium further improved cellulase activity (1.9 FPU per mL). Enzymatic hydrolysis of AFEX-CS using in-house produced enzymes generated high sugar yields with high enzyme loading. Proteomics analyses indicated that EGI and CBHII were deficient in the in-house produced enzyme cocktail when compared to the optimal cocktail for hydrolyzing AFEX-CS.

Sequence-based in silico analysis of well studied Hepatitis C Virus epitopes and their variants in other genotypes (particularly genotype 5a) against South African human leukocyte antigen backgrounds

BackgroundHost genetics influence the outcome of HCV disease. HCV is also highly mutable and escapes host immunity. HCV genotypes are geographically distributed and HCV subtypes have been shown to have distinct repertoires of HLA-restricted viral epitopes which explains the lack of cross protection across genotypes observed in some studies. Despite this, immune databases and putative epitope vaccines concentrate almost exclusively on HCV genotype 1 class I-epitopes restricted by the HLA-A*02 allele. While both genotype and allele predominate in developed countries, we hypothesise that HCV variation and population genetics will affect the efficacy of proposed epitope vaccines in South Africa. This in silico study investigates HCV viral variability within well-studied epitopes identified in genotype 1 and uses algorithms to predict the immunogenicity of their variants from other less studied genotypes and thus rate the most promising vaccine candidates for the South African population. Six class I- and seven class II- restricted epitope sequences within the core, NS3, NS4B and NS5B regions were compared across the six HCV genotypes using local genotype 5a sequence data together with global data. Common HLA alleles in the South African population are A30:01, A02:01, B58:02, B07:02; DRB1*13:01 and DRB1*03:01. Epitope binding to 13 class I- and 8 class –II alleles were described using web-based prediction servers, Immune Epitope Database, (IEDB) and Propred. Online population coverage tools were used to assess vaccine efficacy.ResultsDespite the homogeneity of genotype 1 and genotype 5 over the epitopes, there was limited promiscuity to local HLA-alleles.Host differences will make a putative vaccine less effective in South Africa. Of the 6 well-characterized class I- epitopes, only 2 class I- epitopes were promiscuous and 3 of the 7 class-II epitopes were better conserved and promiscuous. By fine tuning the putative vaccine using an optimal cocktail of genotype 1 and 5a epitopes and local HLA data, the coverage was raised from 65.85% to 91.87% in South African Blacks.ConclusionWhile in vivo and in vitro studies are needed to confirm immunogenic epitopes, in silico HCV epitope vaccine design which takes into account HCV variation and host allele frequency will maximize population coverage in different ethnic groups.

Target-based Anti-angiogenic Therapy in Breast Cancer

Breast cancer is the most common female cancer. Despite advances in prevention, early diagnosis, and surgical treatment, its prognosis is still poor. Therefore, immunological and pharmacological methods of treatment have been emphasized recently. Prominent targets of drugs or antibodies are proteins, which are predominantly expressed on breast cancer cells and are simultaneously involved in promoting cell growth or apoptosis. However, breast cancer is a heterogeneous disease, because breast cancer cells may show a variety of malignant gene expression patterns. Therefore, it is difficult at the current state of technology, to apply the optimal cocktail of drugs to hit all cancer cells of any given patient. Under these circumstances, the option of targeting more tractable, normal cells surrounding the tumor instead of the less heterogeneous ones, preventing them from supporting tumor cell growth, became particularly interesting. Endothelial cells are important supporters of cancer cell growth. As a rule, tumors induce them to grow out and to develop a tumor vasculature, which enables the cancer cells to survive and to spread. The introduction of inhibitors of neovascularization was therefore an important milestone on the way toward treating breast cancer more successfully. It has already been demonstrated that the blockade of vascular endothelial growth results in regression of the disease and first clinical studies seemed to hint toward a beneficial effect on prolongation of survival. Nevertheless, more clinical and basic research is necessary to improve this therapy approach. This review will compile the knowledge about recently developed anti-angiogenic drugs in the treatment of breast cancer and will provide an overview on currently relevant clinical trials.

Pre PCI hospital antithrombotic therapy for ST elevation myocardial infarction: striving for consensus

Strong evidence exists in favor of rapid transfer of a patient suffering an ST-elevation myocardial infarction (STEMI) to the nearest hospital with primary percutaneous coronary intervention (PCI) capability, assuming the time from first medical contact to balloon inflation can be achieved in less than 90 min. In many areas, PCI hospitals have successfully collaborated with regional non-PCI hospitals to provide primary PCI for STEMI; however, significant variations exist in how these programs are executed. For example, the pre PCI hospital administration of antithrombotic agents by emergency medical personnel can include aspirin, clopidogrel, unfractionated heparin, low molecular weight heparin, partial or full dose fibrinolytics or combinations thereof. There is little consensus on the optimal cocktail, dose and route of administration. Standardizing the pre PCI antithrombotic regimen across hospital systems may be one approach to improve timely administration of these therapies, and potentially improve STEMI outcomes.