New technologies for enzyme engineering: Combining computational predictions and automated experimental feedback

The targeted design and optimization of novel enzymes and enzymatic reaction cascades increasingly demands a close connection between rational design, computational prediction and experimental feedback. In recent years, lots of effort have been put on increasing the throughput of experimental results, however, this approach frequently tends to stick in local minima and unsatisfying performance improvement despite considerable screening efforts. Contrary, model-based computational predictions, despite increasing available computation power, need to introduce severe simplifications and therefore will continue to lack accuracy and perfect predictability in the foreseeable future. The interplay of thorough model-based understanding, automated experimental feedback and, based on the latter, refinement of model predictions using for example machine learning methods, will in the near future become an important approach to combine the best of the two worlds. Ultimately, this provides potential to boost highly efficient automated or semi-automated design of new enzymatic properties in the scope of a “fourth wave” of enzyme engineering. We present a new integrated directed evolution framework to achieve this simulation-experimental feedback loop, called “Feedback Guided Enzyme Optimization” (FEO). The implementation includes the setup of a suitable simulation back-end, robot-based experimental generation of mutants and evaluation of their performance [1], and finally feedback to the simulation in order to close the loop and verify and refine the quality of the predictions.Focus is laid on thorough statistical analysis of both prediction and experimental results, in order to tune false positive vs. false negative error rate, depending on experimental conditions: This includes, e.g., availability of time, ingredients, parallel workflows and distortions (random noise and potential systematic deviations) in both experimental and simulation setups. The framework is being implemented in an automated robotic setup. We demonstrate results on three exemplary enzymatic systems: Firstly, GFP is employed as a simple role model to demonstrate the looping principle. The second example, aspartokinase III (AK3), is a key enzyme for the biosynthetic production of amino acids and derivatives thereof. Its activity is naturally limited by its own downstream products, e.g., lysine. Simulated predictions of the sensitivity of AK3 towards lysine have been compared to experimental data. This allowed a significant (p\u3c0.05) simulation-based discrimination of highly resistant versus non-resistant variants. Determination of new lysine resistant mutants by multiple point mutations is performed within few dozen of iterations. The obtained candidates were validated, showing that new Lys-resistant variants can be obtained using the new workflow without special a priori knowledge or extensive (random) screening. The third and most sophisticated enzyme system is the pyruvate dehydrogenase complex (PDC) which involves interesting features like shielding of reaction intermediates, renewal of co-factors, self-assembly, modularity and others. Based on recently published models of PDC by our group [2-3] and in collaborations [4], we demonstrate how the dynamic self-assembly of mutants of PDC and structurally similar enzymes complexes can be predicted, iteratively refined and in the future used for the creation of new enzyme cascades. This presented framework is expected to have large impact on design and evolution of novel biomolecules and biosystems. [1] Wurm, M., Ilhan, S., Jandt, U., Zeng A.-P. (2019). Direct and Highly Sensitive Measurement of Fluorescent Molecules in Bulk Solutions using Flow Cytometry. Analyt. Biochem. 570, 32-42. [2] Hezaveh, S., Zeng, A. P., Jandt, U. (2018). Enzyme Interaction in Human Pyruvate Dehydrogenase Complex: Full Complex Simulation. Journal of Chemical Information and Modeling, 58(2), 362-369. [3] Hezaveh, S., Zeng, A. P., Jandt, U. (2017). Investigation of Core Structure and Stability of Human Pyruvate Dehydrogenase Complex: A Coarse-Grained Approach. ACS Omega, 2(3), 1134-1145. [4] Depta, P.N., Jandt, U., Dosta, M., Zeng, A.-P., Heinrich, S. (2019). Toward Multiscale Modeling of Proteins and Bioagglomerates: An Orientation-Sensitive Diffusion Model for the Integration of Molecular Dynamics and the Discrete Element Method. J. Chem. Inf. Model. 59(1), 386-398

Efficacy and Safety of Prucalopride in Patients with Chronic Noncancer Pain Suffering from Opioid-Induced Constipation

Opioid-induced constipation (OIC) has negative effects on quality of life (QOL). Prucalopride is a new, selective 5-HT4 agonist and enterokinetic with strong clinical data in chronic constipation. This study investigated the efficacy, safety, and tolerability of prucalopride in patients with noncancer pain and OIC. A phase II, double-blind, placebo-controlled study of 196 patients randomized to placebo (n = 66), prucalopride 2 mg (n = 66) or 4 mg (n = 64), for 4 weeks, was carried out. The primary endpoint was the proportion of patients with increase from baseline of a parts per thousand yen1 spontaneous complete bowel movement (SCBM)/week. Secondary endpoints [proportion of patients with a parts per thousand yen3 SCBM/week, weekly frequency of (SC)BM, severity of constipation, and efficacy of treatment], adverse events (AEs), and safety parameters were also monitored. More patients had an increase from baseline of a parts per thousand yen1 SCBM per week (weeks 1-4) in the prucalopride groups [35.9% (2 mg) and 40.3% (4 mg)] versus placebo (23.4%), reaching statistical significance in week 1. Over weeks 1-4, more patients in the prucalopride groups achieved an average of a parts per thousand yen3 SBM per week versus placebo (60.7% and 69.0% versus 43.3%), reaching significance at week 1. Prucalopride 4 mg significantly improved patient-rated severity of constipation and effectiveness of treatment versus placebo. Patient Assessment of Constipation-Symptom (PAC-SYM) total scores and Patient Assessment of Constipation-Quality of Life (PAC-QOL) total and satisfaction subscale scores were improved. The most common AEs were abdominal pain and nausea. There were no clinically relevant differences between groups in vital signs, laboratory measures or electrocardiogram parameters. In this population with OIC, prucalopride improved bowel function and was safe and well tolerated

A Qualitative View of Drug Use Behaviors of Mexican Male Injection Drug Users Deported from the United States

Deportees are a hidden yet highly vulnerable and numerous population. Significantly, little data exists about the substance use and deportation experiences of Mexicans deported from the United States. This pilot qualitative study describes illicit drug use behaviors among 24 Mexico-born male injection drug users (IDUs), ≥18 years old, residing in Tijuana, Mexico who self-identified as deportees from the United States. In-person interviews were conducted in Tijuana, Mexico in 2008. Content analysis of interview transcripts identified major themes in participants’ experiences. Few participants had personal or family exposures to illicit drugs prior to their first U.S. migration. Participants reported numerous deportations. Social (i.e., friends/family, post-migration stressors) and environmental factors (e.g., drug availability) were perceived to contribute to substance use initiation in the U.S. Drugs consumed in the United States included marijuana, heroin, cocaine, methamphetamine, and crack. More than half of men were IDUs prior to deportation. Addiction and justice system experiences reportedly contributed to deportation. After deportation, several men injected new drugs, primarily heroin or methamphetamine, or a combination of both drugs. Many men perceived an increase in their substance use after deportation and reported shame and loss of familial social and economic support. Early intervention is needed to stem illicit drug use in Mexican migrant youths. Binational cooperation around migrant health issues is warranted. Migrant-oriented programs may expand components that address mental health and drug use behaviors in an effort to reduce transmission of blood-borne infections. Special considerations are merited for substance users in correctional systems in the United States and Mexico, as well as substance users in United States immigration detention centers. The health status and health behaviors of deportees are likely to impact receiving Mexican communities. Programs that address health, social, and economic issues may aid deportees in resettling in Mexico

Conservation implications of the mating system of the Pampa Hermosa landrace of peach palm analyzed with microsatellite markers

Peach palm (Bactris gasipaes) is cultivated by many indigenous and traditional communities from Amazonia to Central America for its edible fruits, and is currently important for its heart-of-palm. The objective of this study was to investigate the mating system of peach palm, as this is important for conservation and breeding. Eight microsatellite loci were used to genotype 24 open-pollinated progenies from three populations of the Pampa Hermosa landrace maintained in a progeny trial for genetic improvement. Both the multi-locus outcrossing rates (0.95 to 0.99) and the progeny level multi-locus outcrossing rates (0.9 to 1.0) were high, indicating that peach palm is predominantly allogamous. The outcrossing rates among relatives were significantly different from zero (0.101 to 0.202), providing evidence for considerable biparental inbreeding within populations, probably due to farmers planting seeds of a small number of open-pollinated progenies in the same plot. The correlations of paternity estimates were low (0.051 to 0.112), suggesting a large number of pollen sources (9 to 20) participating in pollination of individual fruit bunches. Effective population size estimates suggest that current germplasm collections are insufficient for long-term ex situ conservation. As with most underutilized crops, on farm conservation is the most important component of an integrated conservation strategy

Documenting the Unauthorized: Political Responses to Unauthorized Immigration

Cultural prejudice rather than self interest is the conventional wisdom for why voters respond negatively to immigration. Using a new measure of unauthorized immigrants based on self-reported invalid social security numbers, we show that voters' responses are more nuanced than mere prejudice against minorities. Using county level data from the U.S. state of Georgia, we find that voters in counties with above median levels of unauthorized workers are more likely to support the Republican party. We also find that wealthier counties and wealthier voters are most likely to respond negatively to the unauthorized. Our evidence warns against arguments that depict opposition to immigration as motivated solely by xenophobia and cultural fears among lower income whites

3D bioactive composite scaffolds for bone tissue engineering

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. Developing bioactive three-dimensional (3D) scaffolds to support bone regeneration has therefore become a key area of focus within bone tissue engineering (BTE). A variety of materials and manufacturing methods including 3D printing have been used to create novel alternatives to traditional bone grafts. However, individual groups of materials including polymers, ceramics and hydrogels have been unable to fully replicate the properties of bone when used alone. Favourable material properties can be combined and bioactivity improved when groups of materials are used together in composite 3D scaffolds. This review will therefore consider the ideal properties of bioactive composite 3D scaffolds and examine recent use of polymers, hydrogels, metals, ceramics and bio-glasses in BTE. Scaffold fabrication methodology, mechanical performance, biocompatibility, bioactivity, and potential clinical translations will be discussed

The disruption of proteostasis in neurodegenerative diseases

Cells count on surveillance systems to monitor and protect the cellular proteome which, besides being highly heterogeneous, is constantly being challenged by intrinsic and environmental factors. In this context, the proteostasis network (PN) is essential to achieve a stable and functional proteome. Disruption of the PN is associated with aging and can lead to and/or potentiate the occurrence of many neurodegenerative diseases (ND). This not only emphasizes the importance of the PN in health span and aging but also how its modulation can be a potential target for intervention and treatment of human diseases.info:eu-repo/semantics/publishedVersio

Real-time Monitoring for the Next Core-Collapse Supernova in JUNO

Core-collapse supernova (CCSN) is one of the most energetic astrophysical events in the Universe. The early and prompt detection of neutrinos before (pre-SN) and during the SN burst is a unique opportunity to realize the multi-messenger observation of the CCSN events. In this work, we describe the monitoring concept and present the sensitivity of the system to the pre-SN and SN neutrinos at the Jiangmen Underground Neutrino Observatory (JUNO), which is a 20 kton liquid scintillator detector under construction in South China. The real-time monitoring system is designed with both the prompt monitors on the electronic board and online monitors at the data acquisition stage, in order to ensure both the alert speed and alert coverage of progenitor stars. By assuming a false alert rate of 1 per year, this monitoring system can be sensitive to the pre-SN neutrinos up to the distance of about 1.6 (0.9) kpc and SN neutrinos up to about 370 (360) kpc for a progenitor mass of 30$M_{\odot}$ for the case of normal (inverted) mass ordering. The pointing ability of the CCSN is evaluated by using the accumulated event anisotropy of the inverse beta decay interactions from pre-SN or SN neutrinos, which, along with the early alert, can play important roles for the followup multi-messenger observations of the next Galactic or nearby extragalactic CCSN.Comment: 24 pages, 9 figure