Upstream Bottleneck Research Articles

Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli

Spider silk proteins (spidroins) are a remarkable class of biomaterials that exhibit a unique combination of high-value attributes and can be processed into numerous morphologies for targeted applications in diverse fields. Recombinant production of spidroins represents the most promising route towards establishing the industrial production of the material, however, recombinant spider silk production suffers from fundamental difficulties that includes low titers, plasmid instability, and translational inefficiencies. In this work, we sought to gain a deeper understanding of upstream bottlenecks that exist in the field through the production of a panel of systematically varied spidroin sequences in multiple E. coli strains. A restriction on basal expression and specific genetic mutations related to stress responses were identified as primary factors that facilitated higher titers of the recombinant silk constructs. Using these findings, a novel strain of E. coli was created that produces recombinant silk constructs at levels 4–33 times higher than standard BL21(DE3). However, these findings did not extend to a similar recombinant protein, an elastin-like peptide. It was found that the recombinant silk proteins, but not the elastin-like peptide, exert toxicity on the E. coli host system, possibly through their high degree of intrinsic disorder. Along with strain engineering, a bioprocess design that utilizes longer culturing times and attenuated induction was found to raise recombinant silk titers by seven-fold and mitigate toxicity. Targeted alteration to the primary sequence of the recombinant silk constructs was also found to mitigate toxicity. These findings identify multiple points of focus for future work seeking to further optimize the recombinant production of silk proteins and is the first work to identify the intrinsic disorder and subsequent toxicity of certain spidroin constructs as a primary factor related to the difficulties of production.

On the Morning Commute Problem in a Y-shaped Network with Individual and Household Travelers

This paper examines the morning commute problem when both household commuters and individual commuters are considered in a Y-shaped network with two upstream links and a single downstream link. The household parents daily pass through an upstream bottleneck with a limited capacity before a school and drop off their children. Then, they traverse the downstream bottleneck common to both household and individual commuters and arrive at the workplace. We explore the effects of staggering policy, that is, staggering work and school start times, on the distribution of traffic congestion and social welfare. We analytically solve all the equilibrium cases and reveal all the traffic congestion patterns. The results reveal that the staggering policy may be harmful in certain cases. When the demand of individuals is relatively low, the staggering policy may not improve social welfare. When the demand of individuals is high, social welfare can be significantly improved if the schedule gap between the work start time and school start time is optimized. The effects of the staggering policy on system performance are examined. We derive a Pareto frontier, which provides a good candidate set for policymakers when the two system performance measures, that is, the total system cost and the total congestion cost, are considered. Our results show that the optimal staggering policy on system performance depends on the demand distribution of the two groups. When the demand of individuals is high, there exists a unique optimal staggering policy that optimizes system performance. However, when the demand of individuals is low, the optimal staggering policy should be selected from the Pareto frontier. Furthermore, we re-examine the capacity expansion paradox under the staggering policy. Our study shows the capacity expansion at the downstream bottleneck can always reduce the total system cost. However, the paradoxical phenomenon may arise when the capacity of the upstream bottleneck is expanded, but it can be eliminated if the schedule gap is properly designed.

Technical Note—A Permutation-Dependent Separability Approach for Capacitated Two-Echelon Inventory Systems

Analyzing Capacitated Two-Echelon Systems with Permutation-Dependent Separability Capacitated multiechelon systems are common in practice due to the escalating costs of labor and advanced manufacturing technology. However, identifying the optimal replenishment policies for such systems is a largely open area of research due to the intrinsic complexity, especially when there is an upstream bottleneck. In “A Permutation-Dependent Separability Approach for Capacitated Two-Echelon Inventory Systems”, Shen, Yu, and Huh propose a new approach, that is, permutation-dependent separability, to tackle a capacitated two-echelon system in which the capacity of upstream stage can be the bottleneck. They show that the value function for the capacitated two-echelon system in each period is permutation-dependent separable, and that for each echelon, a permutation-dependent echelon base stock policy is optimal. The authors also develop efficient solution procedures on how to obtain the optimal policy.

On the morning commute problem in a Y-shaped network with individual and household travelers

This paper examines the morning commute problem when both household commuters and individual commuters are considered in a Y-shaped network with two upstream links and a single downstream link. The household parents daily pass through an upstream bottleneck with a limited capacity before a school and drop off their children. Then, they traverse the downstream bottleneck common to both household and individual commuters and arrive at the workplace. We explore the effects of staggering policy, i.e., staggering work and school start times, on the distribution of traffic congestion and social welfare. We analytically solve all the equilibrium cases and reveal all the traffic congestion patterns. The results reveal that the staggering policy may be harmful in certain cases. When the demand of individuals is relatively low, the staggering policy may not improve social welfare. When the demand of individuals is high, social welfare can be significantly improved if the schedule gap between work start time and school start time is optimized. The effects of the staggering policy on system performance are examined. We derive a Pareto frontier, which provides a good candidate set for the optimal staggering policy. Our results show that the optimal staggering policy on system performance depends on the demand distribution of the two groups. When the demand of individuals is high, there exists a unique optimal staggering policy that minimizes system performance. However, when the demand of individuals is low, the optimal staggering policy should be selected from the Pareto frontier. Furthermore, we re-examine the capacity expansion paradox under the staggering policy. Our study shows the capacity expansion at the downstream bottleneck can always reduce the total system cost. However, the paradoxical phenomenon may arise when the capacity of the upstream bottleneck is expanded, but it can be eliminated if the schedule gap is properly designed.

PS1134 CHRONIC LYMPHOCYTIC LEUKEMIA CELLS IMPAIR MITOCHONDRIAL FITNESS IN T CELLS AND IMPEDE CAR T CELL EFFICACY

Background:Chronic lymphocytic leukemia (CLL) patients acquire T cell dysfunction through a yet unresolved mechanism, impeding efficacy of immunotherapeutic strategies. As T cell function and development share an intricate relationship with T cell metabolism, we hypothesized that CLL cells impose a reduction in mitochondrial fitness and altered glucose metabolism on T cells, which may underlie the acquired T cell dysfunction.Aims:We aim to elucidate the underlying mechanism of T cell dysfunction in CLL and intend to resolve metabolic dysfunction via pharmacological intervention.Methods:We selected untreated CLL patients with a WBC greater than 20 × 109 cells/L. Age‐matched healthy donors (HD) were used as controls. CAR T cells were obtained from the University of Pennsylvania, generated from relapsed/refractory (R/R) CLL patients enrolled in two clinical trials of single‐agent CTL019 therapy (registered at clinicaltrials.gov; NCT01029366, and NCT01747486). We either directly analyzed T cells by flow cytometry or XF96 Seahorse, or after a 2 day culture with or without CD3 and CD28 antibodies.Results:We have previously shown that stimulated CLL derived CD8+ T cells had reduced expression of activation markers, increased expression of PD‐1 and reduced expression of glucose transporter GLUT‐1, coinciding with reduced glucose uptake. In addition we observed that mitochondrial biogenesis is impaired in CLL derived CD8+ T cells. Clinical significance of this data was further demonstrated by analyzing mitochondrial mass of CAR T cells from CLL patients showing complete response (CR) versus patients with no response (NR). Patients with CR had higher mitochondrial mass compared to NR. These findings support our hypothesis that CLL cells reprogram T cell metabolism, in particular mitochondrial fitness and biogenesis (Blood 2018, 132:235).In this follow up study we investigated (A) whether metabolic reprogramming is a pan T cell phenomenon or CD8 specific, (B) upstream signaling leading to metabolic reprogramming, and (C) if pharmacological intervention can boost T cell metabolism and thereby function. We found that (A) CD4 T cells share most metabolic alterations with CD8 T cells in CLL, and that (B) AKT signaling is disturbed in CLL T cells. For the metabolic interventions studies (C) we are currently exploring the effects of manipulating mitochondrial ROS or respiration, and blocking immune‐suppressive cytokines. Results of both approaches will be presented at the conference.Summary/Conclusion:We found that metabolic impairment in CLL T cells is not restricted to CD8 T cells, but also applies to CD4 T cells, and that impaired AKT signaling is at least part of the upstream bottleneck. We are currently investigating the use of compounds and metabolites in order to improve mitochondrial fitness of T cells for CLL patients to benefit autologous immunotherapies.

Exploring the Congestion Pattern at Long-Queued Tunnel Sag and Increasing the Efficiency by Control

The cross-river tunnel is an important facility for modern cities. It is usually a natural bottleneck due to the changes in gradient, which is known as sag. Once a queue propagates from the sag to upstream bottleneck, the induced multi-bottleneck congestion shows more complicated pattern than an isolated one. The lack of understanding on such congestion pattern hampers the application of appropriate control strategies. In this paper, the congestion pattern of the long-queued tunnel sag is investigated at the Xiangyin Tunnel in Shanghai. Three distinctive congestion stages are observed from day-to-day traffic flow data. The mechanisms of congestion are discussed. Based on the understanding of the congestion pattern, a novel control strategy is proposed which cooperatively controls the tunnel sag mainline and the on-ramp. The control objective is to maximize the throughput and minimize the breakdown probability using quantitative risk analysis method. The control strategy is validated in the field experiment. The traffic features (volume, speed, and productivity) in the tunnel sag are found to be significantly enhanced by the control.

Merging Vehicles and Lane Speed-Flow Relationship in a Work Zone

In addition to closed merge lanes as physical bottlenecks of work zones, traffic oscillations caused by merging vehicles at multiple locations could reduce work-zone capacity. This study took a step-wise procedure to reveal spatial distributions of merging vehicles along work zones and their influence on speed-flow relationships of lane traffic flows. Field data showed that inserting vehicles from merge lanes could spread their influence over adjacent unclosed through lanes. Moreover, with increases in total volume, merging vehicles could choose their inserting positions further upstream of the work zone, which could induce oscillations near the insertion point. At the identified upstream bottlenecks, capacity drop was found in speed-flow diagrams of through-lane traffic, but it was not found in the diagrams of merge-lane traffic flows. Lack of sufficient demand and special merging behaviors on merge lanes could be attributed to the distinct speed-flow relationship. Two-part piecewise regression models were developed to fit the speed-flow relationships of uncongested and congested flows of through lanes. By comparing the estimated speed-flow models, it was found that when a queue is forming, the extent of the capacity drop and speed reduction is different for through lanes. Queue discharge uses different lengths of time on through lanes and multiple merging locations.

Efficient genetic approaches for improvement of plasmid based expression of recombinant protein in Escherichia coli : A review

Abstract During the past two decades, there has been an explosion of new knowledge and techniques in the field of recombinant protein expression. However, over-expression of “difficult to express proteins” with therapeutic importance continues to be a challenging task for successful commercialization of these proteins. With the emergence of the bio-similar market, enhancing the efficiencies of the production process has become a critical factor in the commercial viability of novel products. Despite the availability of numerous technological advancements, recombinant protein expression in Escherichia coli remains difficult. Therefore, addressing upstream bottlenecks in combination with genetically modified expression hosts could be a viable strategy to enhance production. Problems like poor expression, plasmid instability, protein aggregation, protein degradation, and metabolic stress associated with recombinant protein production need special consideration during bioprocess development at bioreactor level. However, a comprehensive universal strategy for attaining efficient expression in E. coli seems unrealistic and must be resolved empirically. In this review, we have discussed some common problems and their apparent solutions for plasmids based recombinant gene expression in E. coli.

High-level expression and efficient refolding of therapeutically important recombinant human Interleukin-3 (hIL-3) in E. coli

Human interleukin-3 (hIL-3) is a pleiotropic cytokine that stimulates the differentiation and proliferation of multipotent hematopoietic cells thus making it a therapeutically important molecule. In this study, its poor expression yield was improved by addressing various upstream bottlenecks in E. coli heterologous system. The codon-optimized hIL-3 gene was cloned under various signal sequences and solubility enhancer fusion tags for its hyper-expression under a strong T7 promoter. The optimization of shake flask expression studies resulted in a hIL-3 protein concentration of 225 mg/L in the form of inclusion bodies (IBs). Lowering of inducer concentration and cultivation temperature did not improve its solubility. The hIL-3 protein was refolded from IBs and resulted a protein recovery yield of 53% after optimization of refolding conditions. The refolded protein was subsequently purified using Ni-NTA affinity chromatography and gave ∼95% pure protein. The conformational properties of the refolded hIL-3 protein were studied by CD and fluorescence spectrometry where protein showed 40% α-helix and 12% β-sheets with a fluorescence emission maxima at 344 nm. The molecular identity was further confirmed by MALDI-TOF/TOF and western blot analysis. The biological activity of refolded protein was confirmed via cell proliferation assay on human erythroleukemia TF-1 cells where commercial hIL-3 was taken as a standard control.

Reliable shop floor bottleneck detection for flow lines through process and inventory observations: the bottleneck walk

Bottleneck detection in manufacturing is the key to improving production efficiency and stability in order to improve capacity. Yet, common bottleneck detection methods in industry and academia lack either accuracy or practicability, or both, for dynamic systems. The new methodology is conducted by the observation of processes and inventories. Blocked processes and full inventories indicate a downstream bottleneck. Starved processes and empty inventories indicate an upstream bottleneck. Through subsequent observations of multiple process states and inventory levels within a system, it is possible to determine the direction of the bottleneck at the given time and hence to find the momentary bottleneck in the system. The shifting of bottlenecks can be observed directly. Work sampling techniques can be used to obtain a long-term picture of the dynamically shifting bottleneck. The new methodology does not require any calculations, statistics, or time measurements. Hence, the method is suited for practical use by shop floor supervisors and clerks. The direct observation of the bottleneck also gives additional information about the underlying causes of the bottlenecks, simplifying the improvement of the system capacity. Extensive field testing of the method received positive feedback not only from management but also from shop floor operators. The method is already in use at the Robert Bosch GmbH, where it is known as the bottleneck walk.

Congestion mechanism and demand adjustment strategies for double-cell system with bottlenecks

In this paper, ramp systems on the Beijing 3rd ring road are described as double-cell ramp systems with a bottleneck. By analyzing empirical data for the Beijing 3rd ring road, we found that the initial states have an important impact on the final convergence states of the ramp systems. Then, we studied the dynamic process of the ramp systems, determined the congestion mechanism, and then designed a ramp control method based on the obtained mechanism. Under a feasible demand, double-cell ramp systems exhibit two typical cases, including an upstream-bottleneck system (in which the bottleneck cell is upstream) and a downstream-bottleneck system (in which the bottleneck cell is downstream). Then, a cell transmission model is used to analyze the dynamic evolution processes, starting from different initial states, and determine the congestion mechanism for each case. It is proven that the two systems have different possible equilibrium sets and congestion mechanisms. In an upstream-bottleneck system, the downstream always converges to the uncongested equilibrium, while the upstream bottleneck cell may experience congestion under certain initial states. In a downstream-bottleneck system, the congestion starts downstream, and then gradually propagates upstream. Furthermore, based on the different congestion mechanisms, two demand adjustment strategies are proposed, which redistribute the stationary feasible demand. The simulation results indicate that both systems can converge to uncongested equilibriums after demand adjustment. The ramp demand adjustment methods provide a scientific basis for urban traffic system management.

Quasi-dynamic traffic assignment with residual point queues incorporating a first order node model

Static traffic assignment models are still widely applied for strategic transport planning purposes in spite of the fact that such models produce implausible traffic flows that exceed link capacities and predict incorrect congestion locations. There have been numerous attempts to constrain link flows to capacity. Capacity constrained models with residual queues are often referred to as quasi-dynamic traffic assignment models. After reviewing the literature, we come to the conclusion that an important piece of the puzzle has been missing so far, namely the inclusion of a first order node model. In this paper we propose a novel path-based static traffic assignment model for finding a stochastic user equilibrium in general transportation networks. This model includes a first order (steady-state) node model that yields more realistic turn capacities, which are then used to determine consistent capacity constrained traffic flows, residual point (vertical) queues (upstream bottleneck links), and path travel times consistent with queuing theory. The route choice part of the model is specified as a variational inequality problem, while the network loading part is formulated as a fixed point problem. Both problems are solved using existing techniques to find a solution. We illustrate the model using hypothetical examples, and also demonstrate feasibility on large-scale networks.

Exploring the Influence of the Congested Downstream on Traffic Flow at its Upstream Bottlenecks

With the rapid increase of the private cars, urban road congestion is getting worse. As known, when the downstream section is free flow, the congestion for every bottleneck section starts from the bottleneck cell, and then gradually propagates towards its upstream. Many studies about bottlenecks are based on the assumption that the downstream of each bottleneck is free flow. In this paper, we have investigated ramp systems with the congested downstream. Based on the cell transmission model, a tri-cell ramp system with two bottlenecks is used as an example to explore what the convergence state of the bottleneck section is under the congested downstream section. Three cases have been studied: free-flow downstream, middle-state downstream, and the most congested downstream, and the different convergence states. The results show that 1) if the downstream of the bottleneck is the most congested, the upstream bottleneck section converges to the most congested state no matter what the initial state is; 2) if the downstream is congested, the upstream section converges to congested states, and the degree of congestion in the upstream section increases with; 3) the larger the density in the congested downstream is, the more congested the final state of the ramp system is.

Reliable Shop Floor Bottleneck Detection for Flow Lines through Process and Inventory Observations

Bottleneck detection in manufacturing is the key to improving production efficiency and stability in order to improve capacity. Yet common bottleneck detection methods in industry and academia lack either accuracy or practicability, or both, for dynamic systems. The new methodology is conducted by the observation of processes and inventories. Blocked processes and full inventories indicate a downstream bottleneck. Starved processes and empty inventories indicate an upstream bottleneck. Through subsequent observations of multiple process states and inventory levels within a system, it is possible to determine the direction of the bottleneck at the given time and hence to find the momentary bottleneck in the system. The shifting of bottlenecks can be observed directly. Work-sampling techniques can be used to obtain a long-term picture of the dynamically shifting bottleneck. The new methodology does not require any calculations, statistics, or time measurements. Hence the method is suited for practical use by shop floor supervisors and clerks. The direct observation of the bottleneck also gives additional information about the underlying causes of the bottlenecks, simplifying the improvement of the system capacity. Extensive field testing of the method received positive feedback not only from management but also shop floor operators. The method is already in use at the Robert Bosch GmbH, where it is known as the bottleneck walk.

Regulated Input Price, Vertical Separation, and Leadership in Free Entrymarkets

We examine incentives of bottleneck facility holders to manipulate access charge accounting in free entry downstream markets. We consider the situation wherein one firm holds an upstream bottleneck facility and new entrants use it at the regulated price (access fee) to provide final products. The bottleneck facility holder affects the regulated input price. We investigate how vertical separation affects the incentive for manipulation and the resulting input price. We find that the results depend on whether the incumbent is the Stackelberg leader in the product market. If the incumbent cannot take leadership in the product market and faces Cournot competition, vertical separation reduces the incentive for manipulation and the resulting input price. The opposite result is derived when the incumbent can take leadership in the product market.

System-optimal dynamic traffic assignment with and without queue spillback: Its path-based formulation and solution via approximate path marginal cost

The knowledge of path marginal cost (PMC) is central to system-optimal dynamic traffic assignment (SO-DTA) problems. In this paper, we propose a method to approximate PMC in general networks when traffic dynamics are modeled by either the point-queue or the kinematic wave traffic flow model. This study examines in detail the flow interactions between downstream and upstream bottleneck links, and shows that the changes in cumulative flows on all the network links caused by an arbitrary flow perturbation can be computed. This offers a way to approximate PMC, which is incorporated in the solution of the least marginal cost problem, a central component of the path-based SO-DTA problem. The approximation scheme allows us to solve path-based SO-DTA problems for general networks with and without queue spillback and/or departure time choices. Numerical examples are provided to demonstrate the effectiveness of the proposed method, and the results show that the SO state produces considerably lower total network cost, shorter congestion duration, and smaller travel delay on bottleneck links than those of produced by the user-optimal state, particularly when the departure time choice is considered.

Departure Times in Y-Shaped Traffic Networks with Multiple Bottlenecks

We study the departure time decisions of commuters traversing a traffic network with the goal of arriving at a common destination at a specified time. There are costs associated with arriving either too early or too late, and with delays experienced at bottlenecks. Our main hypothesis, based on the Nash equilibrium distribution of departure times, implies that, for certain parameter values, expanding the capacity of an upstream bottleneck can increase the total travel costs in the network. We report the results of a large-group laboratory experiment, which are strongly supportive of this counterintuitive hypothesis, and we discuss the implications. (JEL D85, R41)

Liberalisation, competition and ownership in the presence of vertical relations

This contribution analyses a market with an upstream bottleneck monopoly and a downstream activity that may either be vertically integrated or separated. Separation always reduces the consumer surplus, and the total surplus unless there are large cost reductions. Downstream competition from a public or private network monopoly would crowd out other firms, also when public ownership is associated with more modest objectives than welfare-maximisation. A market is therefore less likely to remain a mixed oligopoly than without vertical relations. However, private firms would survive in a moderately welfare-improving mixed oligopoly with cross-subsidisation and access charges equal to marginal costs.

Movement Nature of Speed-Flow Relationship on Congested Expressway

The nature of congested traffic flow has been not fully understood until now. An isolated bottleneck on the expressway generally influences a limited segment length, which means attenuation effects of the bottleneck on traffic stream by a distance within the influence area. However, such effects of bottleneck on speed-flow relationship have been inadequately emphasized. This paper investigates the location effects of a bottleneck on the speed-flow relationship. With consecutive 5 weekday median lane data collected by dual loop detectors on a congested expressway in Shanghai, integral speed-flow relationships including upper limb, lower limb, and capacity area are obtained from six sites. Further analyzing those sites on the same basic expressway segment unveils that it is the movement of the upper limb and lower limb that produces the integral curve. The right end of the upper limb gets an increase speed in with the site farther to its upstream bottleneck, whereas the lower limb gets a drop in speed with the site closer to its downstream bottleneck. Such a movement feature is in agreement with general driving behaviors of discharging or approaching a bottleneck. The factors intensifying or weakening the movement tendency are also identified.

ABOLISHING THE PRICE SQUEEZE AS A THEORY OF ANTITRUST LIABILITY

A “price squeeze,” or “margin squeeze,” is a theory of antitrust liability under section 2 of the Sherman Act that concerns a vertically integrated monopolist that sells its upstream bottleneck input to firms that compete with the monopolist's production of a downstream product sold to end users. At issue is the size of the margin between the monopolist's input price and its retail price. Recent antitrust price-squeeze cases have split the U.S. Courts of Appeals. The D.C. Circuit has concluded that, because a vertically integrated monopolist may refuse to provide its upstream inputs to its downstream competitors, it may raise the price of its upstream inputs without incurring antitrust liability. On the other hand, the Ninth Circuit's 2007 linkLine decision rejected such reasoning, notwithstanding Trinko. Predicated on Judge Learned Hand's opinion in Alcoa, linkLine subordinates the protection of consumers to the protection of competitors. It requires access-pricing analysis that more resembles the work of a public utilities commission than that of a federal judge in an antitrust case. Further, the antitrust laws are concerned with the competitive process, not its end results. The inability of a single firm to stay in business is irrelevant as a matter of antitrust law unless the behavior inducing that firm to exit the market also harms the competitive process. The Supreme Court should reverse linkLine and resolve the circuit split. It should revisit Alcoa and explain why alleging a price squeeze neither states a claim in American antitrust law nor justifies deviation from the principles announced in Brooke Group and Trinko.