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To date, much that has been written about ethics and Alzheimer's disease has focused on dilemmas with the central moral framework being some version of principlism. In contrast, this article focuses on the everyday moral questions that relate to essential moral needs--a sense of self that grounds meaning, self-respect, and self-esteem; the quality of the relationship between caregiver and care receiver; and the need to care for the caregiver. A case narrative introduces Mrs. Dodge and her daughter, Linda; an ethical framework that can guide thinking about these everyday concerns follows the narrative. [ABSTRACT FROM AUTHOR]

Background: The focus of this research is to examine the growing use of robotic-assisted minimally invasive esophagectomy. Specifically, it evaluates the immediate clinical and cancer-related results of combining robotic-assisted minimally invasive esophagectomy with a systematic approach to total mesoesophageal excision, as opposed to traditional open transthoracic esophagectomy methods that do not employ a structured total mesoesophageal excision protocol., Methods: A propensity score-matched analysis of 185 robotic-assisted minimally invasive esophagectomies and 223 open transthoracic esophagectomies after standardized Ivor Lewis esophagectomy was performed. After 1:1 nearest neighbor matching to account for confounding by covariates, outcomes of 181 robotic-assisted minimally invasive esophagectomy and 181 open transthoracic esophagectomy were compared., Results: The patient characteristics showed significant differences in the age distribution and in comorbidities such as coronary heart disease, arterial hypertension, and anticoagulant intake. The R0-resection rate of robotic-assisted minimally invasive esophagectomy (96.7%) was significantly higher than open transthoracic esophagectomy (89.0%, P = .004). Thirty-day mortality and hospital mortality showed no significant differences. Postoperative pneumonia rate after robotic-assisted minimally invasive esophagectomy (12.7%) was significantly reduced (open transthoracic esophagectomy 28.7%, P < .001). Robotic-assisted minimally invasive esophagectomy had a significantly shorter intensive care unit stay (P < .001) and shorter hospital stay (P < .001)., Conclusion: This single-center, retrospective study employing propensity score matching found that combining robotic-assisted minimally invasive esophagectomy with structured total mesoesophageal excision results in better short-term clinical and oncologic outcomes than open transthoracic esophagectomy. This finding is significant because the increased rate of R0 resection could indicate a higher likelihood of improved long-term survival. Additionally, enhanced overall postoperative recovery may contribute to better risk management in esophagectomy procedures., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

During biomanufacturing, several unit operations expose solutions of biologics to multiple stresses, such as hydrodynamic shear forces due to fluid flow and interfacial dilatational stresses due to mechanical agitation or bubble collapse. When these stresses individually act on proteins adsorbed to interfaces, it results in an increase in protein particles in the bulk solution, a phenomenon referred to as interface-induced protein particle formation. However, an understanding of the dominant cause, when multiple stresses are acting simultaneously or sequentially, on interface-induced protein particle formation is limited. In this work, we established a unique set-up using a peristaltic pump and a Langmuir-Pockels trough to study the impact of hydrodynamic shear stress due to pumping and interfacial dilatational stress, on protein particle formation. Our experimental results together demonstrate that for protein solutions subjected to various combinations of stress (i.e., interfacial and hydrodynamic stress in different sequences), surface pressure values during adsorption and when subjected to compression/dilatational stresses, showed no change, suggesting that the interfacial properties of the protein film are not impacted by pumping. The concentration of protein particles is an order of magnitude higher when interfacial dilatational stress is applied at the air-liquid interface, compared to solutions that are only subjected to pumping. Furthermore, the order in which these stresses are applied, have a significant impact on the concentration of protein particles measured in the bulk solution. Together, these studies conclude that for biologics exposed to multiple stresses throughout bioprocessing and manufacturing, exposure to air-liquid interfacial dilatational stress is the predominant mechanism impacting protein particle formation at the interface and in the bulk solution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial therapy is a major risk factor for HAP, the underlying mechanism remains incompletely understood. Here, we demonstrate that antibiotic therapy in hospitalized patients is associated with decreased diversity of the gut microbiome and depletion of short-chain fatty acid (SCFA) producers. Infection experiments with mice transplanted with patient fecal material reveal that these antibiotic-induced microbiota perturbations impair pulmonary defense against MDR Klebsiella pneumoniae. This is dependent on inflammatory monocytes (IMs), whose fatty acid receptor (FFAR)2/3-controlled and phagolysosome-dependent antibacterial activity is compromized in mice transplanted with antibiotic-associated patient microbiota. Collectively, we characterize how clinically relevant antibiotics affect antimicrobial defense in the context of human microbiota, and reveal a critical impairment of IM´s antimicrobial activity. Our study provides additional arguments for the rational use of antibiotics and offers mechanistic insights for the development of novel prophylactic strategies to protect high-risk patients from HAP., (© 2024. The Author(s).)

Microphysiological systems (MPSs) are promising in vitro technologies for physiologically relevant predictions of the human absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates. However, polydimethylsiloxane (PDMS), a common material used in MPSs, can both adsorb and absorb small molecules, thereby compromising experimental results. This study aimed to evaluate the feasibility of using the PDMS-based Emulate gut-on-chip to determine the first-pass intestinal drug clearance. In cell-free PDMS organ-chips, we assessed the loss of 17 drugs, among which testosterone was selected as a model compound for further study based on its substantial ad- and absorptions to organ chips and its extensive first-pass intestinal metabolism with well-characterized metabolites. A gut-on-chip model consisting of epithelial Caco-2 cells and primary human umbilical vein endothelial cells (HUVECs) was established. The barrier integrity of the model was tested with reference compounds and inhibition of drug efflux. Concentration-time profiles of testosterone were measured in cell-free organ chips and in gut-on-chip models. A method to deduce the metabolic clearance was provided. Our results demonstrate that metabolic clearance can be determined with PDMS-based MPSs despite substantial compound loss to the chip. Overall, this study offers a practical protocol to experimentally assess ADME properties in PDMS-based MPSs.

The removal of viruses by filtration is a critical unit operation to ensure the overall safety of monoclonal antibody (mAb) products. Many mAbs show very low filtrate flux during virus removal filtration, although there are still significant uncertainties regarding both the mechanisms and antibody properties that determine the filtration behavior. Experiments were performed with three highly purified mAbs through three different commercial virus filters (Viresolve Pro, Viresolve NFP, and Pegasus SV4) with different pore structures and chemistries. The flux decline observed during mAb filtration was largely reversible, even under conditions where the filtrate flux with the mAb was more than 100-fold smaller than the corresponding buffer flux. The extent of flux decline was highly correlated with the hydrodynamic diameter of the mAb as determined by dynamic light scattering (DLS). The mAb with the lowest filtrate flux for all three membranes showed the largest attractive intermolecular interactions and the greatest hydrophobicity, with the latter determined by binding to a butyl resin in an analytical hydrophobic interaction chromatography (HIC) column. These results strongly suggest that the flux behavior is dominated by reversible self-association of the mAbs, providing important insights into the design of more effective virus filtration processes and in the early identification of problematic mAbs/solution conditions., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Biologics manufacturing is capital and consumable intensive with need for advanced inventory planning to account for supply chain constraints. Early-stage process design and technology transfer are often challenging due to limited information on process variability regarding bioreactor titer, process yield, and product quality. Monte Carlo (MC) methods offer a stochastic modeling approach for process optimization where probabilities of occurrence for process inputs are incorporated into a deterministic model to simulate more likely scenarios for process outputs. In this study, we explore MC simulation-based design of a monoclonal antibody downstream manufacturing process. We demonstrate that this probabilistic approach offers more representative outcomes over the conventional worst-case approach where the theoretical minimum and maximum values of each process parameter are used without consideration for their probability of occurrence. Our work demonstrates case studies on more practically sizing unit operations to improve consumable utilization, thereby reducing manufacturing costs. We also used MC simulations to minimize process cadence by constraining the number of cycles per unit operation to fit facility preferences. By factoring in process uncertainty, we have implemented MC simulation-based facility fit analyses to efficiently plan for inventory when accounting for process constraints during technology transfer from lab-scale to clinical or commercial manufacturing., (© 2022 American Institute of Chemical Engineers.)

Detergent-mediated virus inactivation (VI) provides a valuable orthogonal strategy for viral clearance in mammalian processes, in particular for next-generation continuous manufacturing. Furthermore, there exists an industry-wide need to replace the conventionally employed detergent Triton X-100 with eco-friendly alternatives. However, given Triton X-100 has been the gold standard for VI due its minimal impact on protein stability and high inactivation efficacy, inactivation by other eco-friendly detergents and its impact on protein stability is not well understood. In this study, the sugar-based detergent commonly used in membrane protein purification, n-dodecyl-β- d-maltoside was found to be a promising alternative for VI. We investigated a panel of detergents to compare the relative VI efficacy, impact on therapeutic quality attributes, and clearance of the VI agent and other impurities through subsequent chromatographic steps. Detergent-mediated inactivation and protein stability showed comparable trends to low pH inactivation. Using experimental and modeling data, we found detergent-mediated product aggregation and its kinetics to be driven by extrinsic factors such as detergent and protein concentration. Detergent-mediated aggregation was also impacted by an initial aggregation level as well as intrinsic factors such as the protein sequence and detergent hydrophobicity, and critical micelle concentration. Knowledge gained here on factors driving product stability and VI provides valuable insight to design, standardize, and optimize conditions (concentration and duration of inactivation) for screening of detergent-mediated VI., (© 2022 Wiley Periodicals LLC.)

Virus removal filtration is a critical step in the manufacture of monoclonal antibody products, providing a robust size-based removal of both enveloped and non-enveloped viruses. Many monoclonal antibodies show very large reductions in filtrate flux during virus filtration, with the mechanisms governing this behavior and its dependence on the properties of the virus filter and antibody remaining largely unknown. Experiments were performed using the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters using a highly purified monoclonal antibody. The filtrate flux for a 4 g/L antibody solution through the Viresolve® Pro decreased by about 10-fold when the filter was oriented with the skin side down but by more than 1000-fold when the asymmetric filter orientation was reversed and used with the skin side up. The very large flux decline observed with the skin side up could be eliminated by placing a large pore size prefilter directly on top of the virus filter; this improvement in filtrate flux was not seen when the prefilter was used inline or as a batch prefiltration step. The increase in flux due to the prefilter was not related to the removal of large protein aggregates or to an alteration in the extent of concentration polarization. Instead, the prefilter appears to transiently disrupt reversible associations of the antibodies caused by strong intermolecular attractions. These results provide important insights into the role of membrane morphology and antibody properties on the filtrate flux during virus filtration., (© 2022 American Institute of Chemical Engineers.)

Background: Virus inactivation is a critical operation in therapeutic protein manufacturing. Low pH buffers are a widely used strategy to ensure robust enveloped virus clearance. However, the choice of model virus can give varying results in viral clearance studies. Pseudorabies virus (SuHV) or herpes simplex virus-1 (HSV-1) are frequently chosen as model viruses to demonstrate the inactivation for the herpes family., Results: In this study, SuHV, HSV-1, and equine arteritis virus (EAV) were used to compare the inactivation susceptibility at pH 4.0 and 4°C. SuHV and HSV-1 are from the same family, and EAV was chosen as a small, enveloped virus. Glycine, acetate, and citrate buffers at pH 4.0 and varying buffer strengths were studied. The inactivation susceptibility was found to be in the order of SuHV > HSV > EAV. The buffer effectiveness was found to be in the order of citrate > acetate > glycine. The smaller virus, EAV, remained stable and infectious in all the buffer types and compositions studied., Conclusion: The variation in inactivation susceptibility of herpes viruses indicated that SuHV and HSV cannot be interchangeably used as a virus model for inactivation studies. Smaller viruses might remain adventitiously infective at moderately low pH., (© 2021 Wiley-VCH GmbH.)

Viral surrogates to screen for virus inactivation (VI) can be a faster, cheaper and safer alternative to third-party testing of pathogenic BSL2 (Biosafety level 2) model viruses. Although the bacteriophage surrogate, Ø6, has been used to assess low pH BSL2 VI, it has not been used for evaluation of detergent-mediated VI. Furthermore, Ø6 is typically assayed through host cell infectivity which introduces the risk of cross-contaminating other cell lines in the facility. To circumvent contamination, we developed an in-house RT-qPCR (Reverse transcriptase quantitative polymerase chain reaction) assay for selective detection of active Ø6 from a population of live and dead phage. The RT-qPCR assay was used to evaluate Ø6 inactivation in cell culture fluid of monoclonal antibody and fusion protein. Complementary Ø6 infectivity was also conducted at a third-party testing facility. The Ø6 RT-qPCR and infectivity data was modeled against VI of three BSL2 viruses, X- MuLV, A- MuLV and HSV-1 in corresponding therapeutics. Both Ø6 methods demonstrate that any VI agent showing Ø6 clearance of a minimum of 2.5 logs would demonstrate complete BSL2 VI of ≥ 4.0 logs. Compared to BSL2 virus testing, this in-house Ø6 RT-qPCR tool can screen VI agents at 5% the cost and a turnaround time of 2 to 3 days vs. 4 to 7 months., (© 2021 Wiley-VCH GmbH.)

This paper describes a simplified affinity precipitation process for the purification of mAbs from complex mixtures using elastin-like polypeptide fused to a single Z domain of protein A (ELP-Z). This approach eliminates several steps in the original process by directly extracting the mAb from the affinity precipitate, without the need for resolubilization. The efficacy of this elution without resolubilization (EWR) approach for obtaining pure mAb is demonstrated and the effects of mixing are examined. This simplification of the affinity precipitation process may facilitate the implementation of ELP-Z based mAb bioprocessing, particularly in a continuous scenario., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine., Results: Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion., Conclusions: Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane., (© 2021 Wiley-VCH GmbH.)

Process analytical technology (PAT) is a fast-growing field within bioprocessing that enables innovation in biological drug manufacturing. This study demonstrates novel PAT methods for monitoring multiple quality attributes simultaneously during the ultrafiltration and diafiltration (UF/DF) process operation, the final step of monoclonal antibody (mAb) purification. Size exclusion chromatography (SEC) methods were developed to measure excipients arginine, histidine, and high molecular weight (HMW) species using a liquid chromatography (LC) system with autosampler for both on-line and at-line PAT modes. The methods were applied in UF/DF studies for the comparison of single-use tangential flow filtration (TFF) cassettes to standard reusable cassettes to achieve very high concentration mAb drug substance (DS) in the order of 100-200 g/L. These case studies demonstrated that single-use TFF cassettes are a functionally equivalent, low-cost alternative to standard reusable cassettes, and that the on-line PAT measurement of purity and excipient concentration was comparable to orthogonal offline methods. These PAT applications using an on-line LC system equipped with onboard sample dilution can become a platform system for monitoring of multiple attributes over a wide dynamic range, a potentially valuable tool for biological drug development and manufacturing., (© 2021 Wiley Periodicals LLC.)

Charge variants of biological products, such as monoclonal antibodies (mAbs), often play an important role in stability and biological activity. Characterization of these charge variants is challenging, however, primarily due to the lack of both efficient and effective isolation methods. In this work, we present a novel use of an established, high productivity continuous chromatography method, known as multi-column counter-current solvent gradient purification (MCSGP), to create an enriched product that can be better utilized for analytical characterization. We demonstrate the principle of this separation method and compare it to traditional batch HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography) methods, using the isolation of charge variants of different mAbs as a case study. In a majority of cases, we are able to show that the MCSGP method is able to provide enhanced purity and quantity of samples when compared to traditional fractionation methods, using the same separation conditions. In one such case, a sample prepared by MCSGP methodology achieved 95% purity in 10 hours of processing time, while those prepared by FPLC and HPLC achieved purities of 78% and 87% in 48 and 300 hours of processing time, respectively. We further evaluate charge variant enrichment strategies using both salt and pH gradients on cation exchange chromatography (CEX) and anion exchange chromatography (AEX) resins, to provide more effective separation and less sample processing following enrichment. As a result, we find that we are able to utilize different gradients to change the enrichment capabilities of certain charged species. Lastly, we summarize the identified mAb charge variants used in this work, and highlight benefits to analytical characterization of charge variants enriched with the continuous chromatography method. The method adds a new option for charge variant enrichment and facilitates analytical characterization of charge variants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Optimal decision making involving reward uncertainty is integral to adaptive goal-directed behavior. In some instances, these decisions are guided by internal representations of reward history, whereas in other situations, external cues inform a decision maker about how likely certain actions are to yield reward. Different regions of the frontal lobe form distributed networks with striatal and amygdalar regions that facilitate different types of risk/reward decision making. The dorsal medial striatum (DMS) is one key output region of the prefrontal cortex, yet there have been few preclinical studies investigating the involvement of the DMS in different forms of risk/reward decision making. The present study addressed this issue, wherein separate groups of male rats were trained on one of two tasks where they chose between a small/certain or a large/risky reward. In a probabilistic discounting task, reward probabilities changed systematically over blocks of trials (100-6.25% or 6.25-100%), requiring rats to use internal representations of reward history to guide choice. Cue-guided decision-making was assessed with a "Blackjack" task, where different auditory cues indicated the odds associated with the large/risky option (50 or 12.5%). Inactivation of the DMS with GABA agonists impaired adjustments in choice biases during probabilistic discounting, resulting in either increases or decreases in risky choice as the probabilities associated with the large/risky reward decreased or increased over a session. In comparison, DMS inactivation increased risky choices on poor-odds trials on the Blackjack task, which was associated with a reduced impact that non-rewarded choices had on subsequent choices. DMS inactivation also impaired performance of an auditory conditional discrimination. These findings highlight a previously uncharacterized role for the DMS in facilitating flexible action selection during multiple forms of risk/reward decision making., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Beta-glucans are polysaccharides of D-glucose monomers linked by (1-3) beta-glycosidic bonds, are found to have a potential immunogenicity risk in biotherapeutic products, and are labeled as process contaminants. A common source of beta-glucans is from the cellulose found in traditional depth filter media. Typically, beta-glucan impurities that leach into the product from the primary clarification depth filters can be removed by the subsequent bind-and-elute affinity chromatography capture step. Beta-glucans can also be removed by a bind-and-elute cation exchange chromatography step, which is useful for removing beta-glucans introduced by a post-Protein A depth filtration step. However, the increasing prevalence of flowthrough polishing chromatography poses a challenge for beta-glucan removal due to the lack of any bind-and-elute chromatography steps after the post-Protein A depth filter. In this work, a depth filter flush strategy was developed to control beta-glucan leaching into the product pool. Different loading conditions for the depth filtration and subsequent chromatography steps were evaluated to determine the robustness of the optimized flush strategy. Carry through runs demonstrated greater than two-fold reduction in beta-glucan levels using the optimized wash as compared to standard filter flush conditions., (© 2020 American Institute of Chemical Engineers.)

Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid-infrared (Mid-IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid-IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. The platform features integration of fiber optic Mid-IR probe sensors to UF/DF set up at the bulk solution and through a flow cell at the retentate line followed by automated Mid-IR data piping into a process monitoring software platform with pre-loaded partial least square regression (PLS) chemometric models. Data visualization infrastructure is also built-in to the platform so that upon automated PLS prediction of excipients and protein concentrations, the results were projected in a graphical or numerical format in real time. The Mid-IR predicted concentrations of excipients and protein show excellent correlation with the offline measurements by traditional analytical methods. Absolute percent difference values between Mid-IR predicted results and offline reference assay results were ≤5% across all the excipients and the protein of interest; which shows a great promise as a reliable process analytical technology tool., (© 2020 Wiley Periodicals LLC.)

To achieve the high protein concentrations required for subcutaneous administration of biologic therapeutics, numerous manufacturing process challenges are often encountered. From an operational perspective, high protein concentrations result in highly viscous solutions, which can cause pressure increases during ultrafiltration. This can also lead to low flux during ultrafiltration and sterile filtration, resulting in long processing times. In addition, there is a greater risk of product loss from the hold-up volumes during filtration operations. From a formulation perspective, higher protein concentrations present the risk of higher aggregation rates as the closer proximity of the constituent species results in stronger attractive intermolecular interactions and higher frequency of self-association events. There are also challenges in achieving pH and excipient concentration targets in the ultrafiltration/diafiltration (UF/DF) step due to volume exclusion and Donnan equilibrium effects, which are exacerbated at higher protein concentrations. This paper highlights strategies to address these challenges, including the use of viscosity-lowering excipients, appropriate selection of UF/DF cassettes with modified membranes and/or improved flow channel design, and increased understanding of pH and excipient behavior during UF/DF. Additional considerations for high-concentration drug substance manufacturing, such as appearance attributes, stability, and freezing and handling are also discussed. These strategies can be employed to overcome the manufacturing process challenges and streamline process development efforts for high-concentration drug substance manufacturing., (© 2020 Wiley Periodicals LLC.)

Maladaptive decision making is a characteristic feature of substance use disorder and pathological gambling. Studies in humans and animals have implicated neural circuits that include the basolateral amygdala (BLA) and nucleus accumbens (NAc) in facilitating risk/reward decision making. However, the preclinical literature has focussed primarily on situations where animals use internally-generated information to adapt to changes in reward likelihood, whereas many real-life situations require the use of external stimuli to facilitate context-appropriate behavior. We recently developed the "Blackjack" task, to measure cued risk/reward decision making requiring rats to chose between Small/Certain and Large/Risky rewards, with auditory cues at the start of each trial explicitly informing that the probability of obtaining a large reward was either good (50%) or poor (12.5%). Here we investigated the contribution of the BLA and its interaction with the NAc in guiding these types of decisions. In well-trained male rats, bilateral inactivation of the BLA induced suboptimal decision making, primarily by reducing risky choice on good-odds trials. In comparison, pharmacological disconnection of the BLA and NAc-shell also induced suboptimal decision making, diverting choice from more preferred option by reducing or increasing risky choice on good vs. poor odds trials respectively. Together, these results suggest that the BLA-NAc circuitry plays a crucial role in integrating information provided by discriminative stimuli. Furthermore, this circuitry may aid in guiding action selection of advantageous options in situations to maximize rewards. Finally, they suggest that perturbations in optimal decision making observed in substance abuse and gambling disorders may be driven in part by dysfunction within this circuitry., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Converging evidence from studies with animals and humans have implicated separate regions of the medial prefrontal cortex (mPFC) corresponding to the anterior cingulate cortex (ACC), in mediating different aspects of reward-related decisions involving uncertainty or risk. However, the dissociable contributions of subregions of the ACC remain unclear, as discrepancies exist between human neuroimaging findings and preclinical rodent studies. To clarify how ventral vs. dorsal regions of the mPFC contribute to risk/reward decision making, the present study assessed the effects of inactivation of different subregions on performance of a "Blackjack task" that measured cue-guided decision making and shares similarities with paradigms used with humans. Male, Long-Evans rats were well-trained to choose between a Small/Certain reward vs a Large/Risky reward delivered with variable probabilities (i.e., good vs. poor-odds, 50% vs. 12.5%). The odds of obtaining the larger reward was signaled by auditory cues at the start of each trial. Inactivation of the ventral, infralimbic region of the mPFC increased risky choice selectively when the odds of winning were poor. By contrast, inactivation of the prelimbic and anterior cingulate regions of the dorsal mPFC led to suboptimal reductions in risky choice on good-odds trials. The effects of prelimbic vs anterior cingulate inactivations were associated with context-dependent alterations in reward vs negative feedback, respectively. These results further clarify the distinct yet complementary manners in which separate ACC regions promote optimal risk/reward decision making and complement neuroimaging findings that activity in human ventral vs dorsal ACC promotes risk aversion or risky choices.

Acute stress and corticotropin-releasing factor (CRF) have been show to perturb cost/benefit decision making involving effort costs. However, previous studies on how stress manipulations affect decisions involving reward uncertainty have yielded variable results. To provide additional insight into this issue, the current study investigated how central CRF infusion and acute restraint stress alter different forms of risk/reward decision-making guided by internal representations of risk/reward contingencies or external informative cues. Male rats were well-trained on one of two tasks that required choice between a small/certain or a large/risky reward. On a probabilistic discounting task, the probability of obtaining the larger reward increased or decreased systematically over blocks of trials (100-6.25%). On a cue-guided Blackjack task, reward probabilities (50% or 12.5%) were signaled by discriminative auditory cues. CRF (1 or 3 μg) was infused intracerebroventricularly (ICV) or one-hour of restraint stress was administered prior to behavioral testing. Neither CRF nor acute stress altered risky choice on probabilistic discounting, but did increase trial omissions in the latter part of the session. Conversely on the Blackjack task, CRF reduced risky choice on good-odds trials (50%), whereas acute stress increased reward sensitivity. CRF but not acute stress also slowed decision latencies across tasks. These data reveal complex and differential manners in which increased CRF activity and acute stress alter distinct forms of risk/reward decision-making, particularly those guided by external cues., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Label-free in vitro potency assays are an emerging field in drug discovery to enable more physiological conditions, to improve the readout quality, and to save time. For this approach mass spectrometry (MS) is a powerful technology to directly follow physiological processes. The speed of this methodology, however, was for a long time not compatible with chemiluminescence- or fluorescence-based assays. Recent advances in matrix-assisted laser desorption/ionization (MALDI) instrumentation paved the way for high-throughput MS analysis of label-free assays for large compound libraries, whereas electrospray ionization (ESI)-based mass spectrometers equipped with RapidFire autosamplers were limited to medium throughput. Here we present a technological advancement of the RapidFire device to enable cycle times of 2.5 s per sample. This newly developed BLAZE-mode substantially boosted the ESI-MS analysis speed, providing an alternative technology for label-free high-throughput screening.

Viral inactivation plays a critical role in assuring the safety of monoclonal antibody (mAb) therapeutics. Traditional viral inactivation involves large holding tanks in which product is maintained at a target low pH for a defined hold time, typically 30-60 min. The drive toward continuous processing and improved facility utilization has provided motivation for development of a continuous viral inactivation process. To this end, a lab-scale prototype viral inactivation system was designed, built, and characterized. Multiple incubation chamber designs are evaluated to identify the optimal design that enables narrow residence time distributions in continuous flow systems. Extensive analysis is conducted supporting rapid low pH viral inactivation and included evaluations with multiple viruses, a range of pH levels, buffer compositions, mAb concentrations, and temperatures. Multiple test conditions are evaluated using the in-line system and results compared to traditional batch-mode viral inactivation. Comparability in kinetics of virus inactivation suggests equivalency between the two approaches., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34 + cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34 + cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34 + cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)