The Tandem CARDs of NOD2: Intramolecular Interactions and Recognition of RIP2

Caspase recruitment domains (CARDs) are homotypic protein interaction modules that link the stimulus-dependent assembly of large signaling platforms such as inflammasomes to the activation of downstream effectors that often include caspases and kinases and thereby play an important role in the regulation of inflammatory and apoptotic signaling pathways. NOD2 belongs to the NOD-like (NLR) family of intracellular pattern recognition receptors (PRR) and induces activation of the NF-κB pathway in response to the recognition of bacterial components. This process requires the specific recognition of the CARD of the protein kinase RIP2 by the tandem CARDs of NOD2. Here we demonstrate that the tandem CARDs of NOD2 are engaged in an intramolecular interaction that is important for the structural stability of this region. Using a combination of ITC and pull-down experiments we identify distinct surface areas that are involved in the intramolecular tandem CARD interaction and the interaction with the downstream effector RIP2. Our findings indicate that while CARDa of NOD2 might be the primary binding partner of RIP2 the two CARDs of NOD2 do not act independently of one another but may cooperate to from a binding surface that is distinct from that of single CARDs

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies

The effect on endothelial function of vitamin C during methionine induced hyperhomocysteinaemia

BACKGROUND: Manipulation of total homocysteine concentration with oral methionine is associated with impairment of endothelial-dependent vasodilation. This may be caused by increased oxidative stress. Vitamin C is an aqueous phase antioxidant vitamin and free radical scavenger. We hypothesised that if the impairment of endothelial function related to experimental hyperhomocysteinaemia was free radically mediated then co-administration of vitamin C should prevent this. METHODS: Ten healthy adults took part in this crossover study. Endothelial function was determined by measuring forearm blood flow (FBF) in response to intra-arterial infusion of acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). Subjects received methionine (100 mg/Kg) plus placebo tablets, methionine plus vitamin C (2 g orally) or placebo drink plus placebo tablets. Study drugs were administered at 9 am on each study date, a minimum of two weeks passed between each study. Homocysteine (tHcy) concentration was determined at baseline and after 4 hours. Endothelial function was determined at 4 hours. Responses to the vasoactive substances are expressed as the area under the curve of change in FBF from baseline. Data are mean plus 95% Confidence Intervals. RESULTS: Following oral methionine tHcy concentration increased significantly versus placebo. At this time endothelial-dependent responses were significantly reduced compared to placebo (31.2 units [22.1-40.3] vs. 46.4 units [42.0-50.8], p < 0.05 vs. Placebo). Endothelial-independent responses were unchanged. Co-administration of vitamin C did not alter the increase in homocysteine or prevent the impairment of endothelial-dependent responses (31.4 [19.5-43.3] vs. 46.4 units [42.0-50.8], p < 0.05 vs. Placebo) CONCLUSIONS: This study demonstrates that methionine increased tHcy with impairment of the endothelial-dependent vasomotor responses. Administration of vitamin C did not prevent this impairment and our results do not support the hypothesis that the endothelial impairment is mediated by adverse oxidative stress

The Incidence of Adjacent Segment Degeneration after Cervical Disc Arthroplasty (CDA): A Meta Analysis of Randomized Controlled Trials

Cervical disc arthroplasty is being used as an alternative degenerative disc disease treatment with fusion of the cervical spine in order to preserve motion. However, whether replacement arthoplasty in the spine achieves its primary patient centered objective of lowering the frequency of adjacent segment degeneration is not verified yet.We conducted a meta-analysis according to the guidelines of the Cochrane Collaboration using databases including PubMed, Cochrane Central Register of Controlled Trials and Embase. The inclusion criteria were: 1) Randomized, controlled study of degenerative disc disease of the cervical spine involving single segment or double segments using Cervical disc arthroplasty (CDA) with anterior cervical discectomy and fusion (ACDF) as controls; 2) A minimum of two-year follow-up using imaging and clinical analyses; 3) Definite diagnostic evidences for "adjacent segment degeneration" and "adjacent segment disease"; 4) At least a minimum of 30 patients per population. Two authors independently selected trials; assessed methodological quality, extracted data and the results were pooled.No study has specifically compared the results of adjacent segment degenerative; Two papers describing 140 patients with 162 symptomatic cervical segment disorders and compared the rate of postoperative adjacent segment disease development between CDA and ACDF treatments, three publications describing the rate of adjacent-segment surgery including 1273 patients with symptomatic cervical segments. The result of the meta-analysis indicates that there were fewer the rate of adjacent segment disease and the rate for adjacent-segment surgery comparing CDA with ACDF, but the difference was not statistically significant.Based on available evidence, it cannot be concluded, that CDA can significantly reduce the postoperative rate of the adjacent segment degenerative and adjacent segment disease. However, due to some limitations, the results of this meta-analysis should be cautiously accepted, and further studies are needed

Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway

Dual Beneficial Effects of (-)-Epigallocatechin-3-Gallate on Levodopa Methylation and Hippocampal Neurodegeneration: In Vitro and In Vivo Studies

A combination of levodopa (L-DOPA) and carbidopa is the most commonly-used treatment for symptom management in Parkinson's disease. Studies have shown that concomitant use of a COMT inhibitor is highly beneficial in controlling the wearing-off phenomenon by improving L-DOPA bioavailability as well as brain entry. The present study sought to determine whether (-)-epigallocatechin-3-gallate (EGCG), a common tea polyphenol, can serve as a naturally-occurring COMT inhibitor that also possesses neuroprotective actions.Using both in vitro and in vivo models, we investigated the modulating effects of EGCG on L-DOPA methylation as well as on chemically induced oxidative neuronal damage and degeneration. EGCG strongly inhibited human liver COMT-mediated O-methylation of L-DOPA in a concentration-dependent manner in vitro, with an average IC50 of 0.36 microM. Oral administration of EGCG moderately lowered the accumulation of 3-O-methyldopa in the plasma and striatum of rats treated with L-DOPA+carbidopa. In addition, EGCG also reduced glutamate-induced oxidative cytotoxicity in cultured HT22 mouse hippocampal neuronal cells through inactivation of the nuclear factor kappaB-signaling pathway. Under in vivo conditions, administration of EGCG exerted a strong protective effect against kainic acid-induced oxidative neuronal death in the hippocampus of rats.These observations suggest that oral administration of EGCG may have significant beneficial effects in Parkinson's patients treated with L-DOPA and carbidopa by exerting a modest inhibition of L-DOPA methylation plus a strong neuroprotection against oxidative damage and degeneration

Down-Regulation of GEP100 Causes Increase in E-Cadherin Levels and Inhibits Pancreatic Cancer Cell Invasion

AIMS: Invasion and metastasis are major reasons for pancreatic cancer death and identifying signaling molecules that are specifically used in tumor invasion is of great significance. The purpose of this study was to elucidate the role of GEP100 in pancreatic cancer cell invasion and metastasis and the corresponding molecular mechanism. METHODS: Stable cell lines with GEP100 knocked-down were established by transfecting GEP100 shRNA vector into PaTu8988 cells and selected by puromycin. qRT-PCR and Western blot were performed to detect gene expression. Matrigel-invasion assay was used to detect cancer cell invasion in vitro. Liver metastasis in vivo was determined by splenic injection of indicated cell lines followed by spleen resection. Immunofluorescence study was used to detect the intracellular localization of E-cadherin. RESULTS: We found that the expression level of GEP100 protein was closely related to the invasive ability of a panel of 6 different human pancreatic cancer cell lines. Down-regulation of GEP100 in PaTu8988 cells significantly decreased invasive activity by Matrigel invasion assay, without affecting migration, invasion and viability. The inhibited invasive activity was rescued by over-expression of GEP100 cDNA. In vivo study showed that liver metastasis was significantly decreased in the PaTu8988 cells with GEP100 stably knocked-down. In addition, an epithelial-like morphological change, mimicking a mesenchymal to epithelial transition (MET) was induced by GEP100 down-regulation. The expression of E-cadherin protein was increased 2-3 folds accompanied by its redistribution to the cell-cell contacts, while no obvious changes were observed for E-cadherin mRNA. Unexpectedly, the mRNA of Slug was increased by GEP100 knock-down. CONCLUSION: These findings provided important evidence that GEP100 plays a significant role in pancreatic cancer invasion through regulating the expression of E-cadherin and the process of MET, indicating the possibility of it becoming a potential therapeutic target against pancreatic cancer

Antimicrobial Properties of Ethylene Vinyl Alcohol/Epsilon-Polylysine Films and Their Application in Surimi Preservation

[EN] Polymer films based on ethylene vinyl copolymers (EVOH) containing a 29 % (EVOH 29) and a 44 % molar percentage of ethylene (EVOH 44), and incorporating epsilon-polylysine (EPL) at 0 %, 1 %, 5 % and 10 % were successfully made by casting. The optical properties and the amount of EPL released from the films to phosphate buffer at pH 7.5 were evaluated, films showing great transparency and those of EVOH 29 copolymer releasing a greater amount of EPL. The antimicrobial properties of the resulting films were tested in vitro against different foodborne microorganisms and in vivo in surimi sticks. With regard to the antimicrobial capacity tested in vitro in liquid medium at 37 A degrees C and 4 A degrees C against Listeria monocytogenes and Escherichia coli over a period of 72 h, films showed a considerable growth inhibitory effect against both pathogens, more notably against L. monocytogenes, and being EVOH 29 more effective than EVOH 44 films. At 37 A degrees C, total growth inhibition was observed for EVOH 29 films incorporating 10 % EPL against both microorganisms whereas the copolymer EVOH 44 did show total inhibition against L. monocytogenes and the growth of E. coli was reduced by 6.64 log units. At 4 A degrees C, no film was able to inhibit completely bacterial growth. Scanning electron microscopy micrographs showed corrugated cell surfaces with blisters and bubbles, and collapse of the cells appearing shorter and more compact after treatment with EPL. Finally, the films were successfully used to increase the shelf life of surimi sticks. The results show the films developed have a great potential for active food packaging applications.The authors acknowledge the financial support of the Spanish Ministry of Economy and Competitiveness, projects AGL2012-39920-C03-01, and fellowship funding for V. M.-G.Muriel-Galet, V.; Lopez-Carballo, G.; Gavara Clemente, R.; Hernández-Muñoz, P. (2014). Antimicrobial Properties of Ethylene Vinyl Alcohol/Epsilon-Polylysine Films and Their Application in Surimi Preservation. Food and Bioprocess Technology. 7(12):3548-3559. https://doi.org/10.1007/s11947-014-1363-1S35483559712Adams, M. R., & Moss, M. O. (2008). Food microbiology. UK: The Royal Society of Chemistry Cambridge.Aucejo, S., Catala, R., & Gavara, R. (2000). Interactions between water and EVOH food packaging films. Food Science and Technology International, 6(2), 159–164.Brandt, A. L., Castillo, A., Harris, K. B., Keeton, J. T., Hardin, M. D., & Taylor, T. M. (2010). Inhibition of Listeria monocytogenes by food antimicrobials applied singly and in combination. Journal of Food Science, 75(9), 557–563.Buchanan, R. L., & Doyle, M. P. (1997). Foodborne disease significance of Escherichia coli O157:H7 and other enterohemorrhagic E-coli. Food Technology, 51(10), 69–76.Chang, S.-S., Lu, W.-Y. W., Park, S.-H., & Kang, D.-H. (2010). Control of foodborne pathogens on ready-to-eat roast beef slurry by epsilon-polylysine. International Journal of Food Microbiology, 141(3), 236–241.Chang, Y., McLandsborough, L., & McClements, D. J. (2012). Cationic antimicrobial (epsilon-polylysine)-anionic polysaccharide (Pectin) interactions: influence of polymer charge on physical stability and antimicrobial efficacy. Journal of Agricultural and Food Chemistry, 60(7), 1837–1844.Chi-Zhang, Y. D., Yam, K. L., & Chikindas, M. L. (2004). Effective control of Listeria monocytogenes by combination of nisin formulated and slowly released into a broth system. International Journal of Food Microbiology, 90(1), 15–22.Coton, M., Denis, C., Cadot, P., & Coton, E. (2011). Biodiversity and characterization of aerobic spore-forming bacteria in surimi seafood products. Food Microbiology, 28(2), 252–260.FAO (2005) Further processing of fish Fisheries and Aquaculture Department, Rome. Updated 27 May 2005 Retrieved 14 March 2011.FDA (2004) Agency reponse letter GRAS Notice No. GRN 00135.Gambarin, P., Magnabosco, C., Losio, M. N., Pavoni, E., Gattuso, A., Arcangeli, G., et al. (2012). Listeria monocytogenes in ready-to-rat seafood and potential hazards for the consumers. International Journal of Microbiology, 2012, 497–635.Geornaras I, Yoon Y., Belk K. E., Smith G. C., Sofos J. N. (2007). Antimicrobial activity of epsilonpolylysine against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in various food extracts. Journal of Food Science, 72(8), M330–4.Gunlu, A., & Koyun, E. (2013). Effects of vacuum packaging and wrapping with chitosan-based edible film on the extension of the shelf life of sea bass (Dicentrarchus labrax) fillets in cold storage (4 A degrees C). Food and Bioprocess Technology, 6(7), 1713–1719.Hiraki, J. (1995). Basic and applied studies on ε-polylysine. Journal of Antibacterial Antifungal Agents Japan, 23, 349–493.Hiraki, J. (2000). ε-Polylysine, its development and utilization. Fine Chemistry, 29, 18–25.Hiraki, J., Ichikawa, T., Ninomiya, S., Seki, H., Uohama, K., Kimura, S., et al. (2003). Use of ADME studies to confirm the safety of epsilon-polylysine as a preservative in food. Regulatory Toxicology and Pharmacology, 37(2), 328–340.Ho, Y. T., Ishizaki, S., & Tanaka, M. (2000). Improving emulsifying activity of epsilon-polylysine by conjugation with dextran through the Maillard reaction. Food Chemistry, 68(4), 449–455.Huss, H. H., Jorgensen, L. V., & Vogel, B. F. (2000). Control options for Listeria monocytogenes in seafoods. International Journal of Food Microbiology, 62(3), 267–274.Kaneko, K., Hayashidani, H., Ohtomo, Y., Kosuge, J., Kato, M., Takahashi, K., et al. (1999). Bacterial contamination of ready-to-eat foods and fresh products in retail shops and food factories. Journal of Food Protection, 62(6), 644–649.Kang, E. T., Tan, K. L., Kato, K., Uyama, Y., & Ikada, Y. (1996). Surface modification and functionalization of polytetrafluoroethylene films. Macromolecules, 29(21), 6872–6879.Li, J., Han, Q., Chen, W., & Ye, L. (2012). Antimicrobial activity of Chinese bayberry extract for the preservation of surimi. Journal of the Science of Food and Agriculture, 92(11), 2358–2365.Lopez de Dicastillo, C., Nerin, C., Alfaro, P., Catala, R., Gavara, R., & Hernandez-Munoz, P. (2011). Development of new antioxidant active packaging films based on ethylene vinyl alcohol copolymer (EVOH) and green tea extract. Journal of Agricultural and Food Chemistry, 59(14), 7832–7840.Lopez-de-Dicastillo, C., Alonso, J. M., Catala, R., Gavara, R., & Hernandez-Munoz, P. (2010). Improving the antioxidant protection of packaged food by incorporating natural flavonoids into ethylene-vinyl alcohol copolymer (EVOH) dilms. Journal of Agricultural and Food Chemistry, 58(20), 10958–10964.Lopez-de-Dicastillo, C., Pezo, D., Nerin, C., Lopez-Carballo, G., Catala, R., Gavara, R., et al. (2012). Reducing oxidation of foods through antioxidant active packaging based on ethyl vinyl alcohol and natural flavonoids. Packaging Technology and Science, 25(8), 457–466.M100-S22 (2012) Performance Standards for Antimicrobial Susceptibility Testing: Eighteenth Informational Supplement. Clinical and Laboratory Standards Institute. Advancing Quality in Health Care Testing. Vol. 32 No. 3. Replaces M100-S21 . Vol. 31 No. 1Mead, P. S., & Griffin, P. M. (1998). Escherichia coli O157:H7. Lancet, 352(9135), 1207–1212.Miya, S., Takahashi, H., Ishikawa, T., Fujii, T., & Kimura, B. (2010). Risk of Listeria monocytogenes xontamination of raw ready-to-eat seafood products available at retail outlets in Japan. Applied and Environmental Microbiology, 76(10), 3383–3386.Muriel-Galet, V., Cerisuelo, J. P., Lopez-Carballo, G., Lara, M., Gavara, R., & Hernandez-Munoz, P. (2012a). Development of antimicrobial films for microbiological control of packaged salad. International Journal of Food Microbiology, 157(2), 195–201.Muriel-Galet, V., Lopez-Carballo, G., Gavara, R., & Hernandez-Munoz, P. (2012b). Antimicrobial food packaging film based on the release of LAE from EVOH. International Journal of Food Microbiology, 157(2), 239–244.Muriel-Galet, V., Cerisuelo, J. P., Lopez-Carballo, G., Aucejo, S., Gavara, R., & Hernandez-Munoz, P. (2013a). Evaluation of EVOH-coated PP films with oregano essential oil and citral to improve the shelf-life of packaged salad. Food Control, 30(1), 137–143.Muriel-Galet, V., López-Carballo, G., Hernández-Muñoz, P., & Gavara, R. (2013b). Characterization of ethylene–vinyl alcohol copolymer containing lauril arginate (LAE) as material for active antimicrobial food packaging. Food Packaging and Shelf Life, 1, 10–17.Park, J. W. (2014). Surimi and surimi seafood. Boca Raton: CRC Press.Shima, S., & Sakai, H. (1977). Polylysine produced by Streptomyces. Agricultural and Biological Chemistry, 41(9), 1807–1809.Shima, S., Matsuoka, H., Iwamoto, T., & Sakai, H. (1984). Antimicrobial action of epsilon-poly-l-lysine. Journal of Antibiotics, 37(11), 1449–1455.Singh, R. K., & Balange, A. K. (2005). Characteristics of pink perch (Nemipterus japonicus) surimi at frozen temperature. Journal of Food Processing and Preservation, 29(1), 75–83.Suppakul, P., Miltz, J., Sonneveld, K., & Bigger, S. W. (2003). Active packaging technologies with an emphasis on antimicrobial packaging and its applications. Journal of Food Science, 68(2), 408–420.Ting, H. Y., Ishizaki, S., & Tanaka, M. (1999). Epsilon-polylysine improves the quality of surimi products. Journal of Muscle Foods, 10(4), 279–294.Tzschoppe, M., Martin, A., & Beutin, L. (2012). A rapid procedure for the detection and isolation of enterohaemorrhagic Escherichia coli (EHEC) serogroup O26, O103, O111, O118, O121, O145 and O157 strains and the aggregative EHEC O104:H4 strain from ready-to-eat vegetables. International Journal of Food Microbiology, 152(1–2), 19–30.Uchida, E., Uyama, Y., & Ikada, Y. (1993). Sorption of low-molecular-weight anions into thin polycation layers grafted onto a film. Langmuir, 9(4), 1121–1124.Unalan, I. U., Ucar, K. D. A., Arcan, I., Korel, F., & Yemenicioglu, A. (2011). Antimicrobial potential of polylysine in edible films. Food Science and Technology Research, 17(4), 375–380.Venugopal, V., & Shahidi, F. (1995). Value-added products from underutilized fish species. Critical Reviews in Food Science and Nutrition, 35(5), 431–453.Zambuchini, B., Fiorini, D., Verdenelli, M. C., Orpianesi, C., & Ballini, R. (2008). Inhibition of microbiological activity during sole (Solea solea L.) chilled storage by applying ellagic and ascorbic acids. LWT--Food Science and Technology, 41(9), 1733–1738.Zinoviadou, K. G., Koutsoumanis, K. P., & Biliaderis, C. G. (2010). Physical and thermo-mechanical properties of whey protein isolate films containing antimicrobials, and their effect against spoilage flora of fresh beef. Food Hydrocolloids, 24(1), 49–59

An evaluation of three DoE-guided meta-heuristic-based solution methods for a three-echelon sustainable distribution network

This article evaluates the efficiency of three meta-heuristic optimiser (viz. MOGA-II, MOPSO and NSGA-II)-based solution methods for designing a sustainable three-echelon distribution network. The distribution network employs a bi-objective location-routing model. Due to the mathematically NP-hard nature of the model a multi-disciplinary optimisation commercial platform, modeFRONTIER®, is adopted to utilise the solution methods. The proposed Design of Experiment (DoE)-guided solution methods are of two phased that solve the NP-hard model to attain minimal total costs and total CO2 emission from transportation. Convergence of the optimisers are tested and compared. Ranking of the realistic results are examined using Pareto frontiers and the Technique for Order Preference by Similarity to Ideal Solution approach, followed by determination of the optimal transportation routes. A case of an Irish dairy processing industry’s three-echelon logistics network is considered to validate the solution methods. The results obtained through the proposed methods provide information on open/closed distribution centres (DCs), vehicle routing patterns connecting plants to DCs, open DCs to retailers and retailers to retailers, and number of trucks required in each route to transport the products. It is found that the DoE-guided NSGA-II optimiser based solution is more efficient when compared with the DoE-guided MOGA-II and MOPSO optimiser based solution methods in solving the bi-objective NP-hard three-echelon sustainable model. This efficient solution method enable managers to structure the physical distribution network on the demand side of a logistics network, minimising total cost and total CO2 emission from transportation while satisfying all operational constraints