Engineering Efforts Research Articles

Modulation of AAV9 Galactose Binding Yields Novel Gene Therapy Vectors and Predicts Cross-Species Differences in Glycan Avidity.

Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extrahepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect invivo AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a 6-fold reduction in liver RNA expression and a 10-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extrahepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies.

Embedded ML-based locomotion control for a 12-joint four-legged robot

Reinforcement learning has developed as a promising approach for robot locomotion control, which can save engineering effort compared to conventional approaches. This article presents the implementation of Reinforcement learning on a low-cost, 12 degree of freedom robot known as a quadruped (spider) to optimize locomotion control, enabling the robot to move on different surfaces such as flat surfaces, ramps, speed bumps, and rough terrain. A MATLAB Simulink model is developed as a digital twin of the spider robot. The dynamics of the model were studied and validated using an open-loop algorithm. Then, the model is utilized in a training simulation environment to apply the reinforcement learning algorithm, showing its ability to move along a predefined path as a replacement for conventional motion control systems. Moreover, the work compares the performance and effectiveness of machine learning-based locomotion control with traditional motion control systems regarding navigation accuracy, speed, and adaptability in challenging environments. The minimum hardware requirements are also studied to move the experiment from simulation to reality.

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is CHMS dehydrogenase, which acts on the substrate 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS). We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme’s mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

Software stewardship and advancement of a high-performance computing scientific application: QMCPACK

We provide an overview of the software engineering efforts and their impact in QMCPACK, a production-level ab-initio Quantum Monte Carlo open-source code targeting high-performance computing (HPC) systems. Aspects included are: (i) strategic expansion of continuous integration (CI) targeting CPUs, using GitHub Actions own runners, and NVIDIA and AMD GPUs used in pre-exascale systems, (ii) incremental reduction of memory leaks using sanitizers, (iii) incorporation of Docker containers for CI and reproducibility, and (iv) refactoring efforts to improve maintainability, testing coverage, and memory lifetime management. We quantify the value of these improvements by providing metrics to illustrate the shift towards a predictive, rather than reactive, maintenance approach. Our goal, in documenting the impact of these efforts on QMCPACK, is to contribute to the body of knowledge on the importance of research software engineering (RSE) for the stewardship and advancement of community HPC codes to enable scientific discovery at scale.

CYP76BK1 orthologs catalyze furan and lactone ring formation in clerodane diterpenoids across the mint family.

The Lamiaceae (mint family) is the largest known source of furanoclerodanes, a subset of clerodane diterpenoids with broad bioactivities including insect antifeedant properties. The Ajugoideae subfamily, in particular, accumulates significant numbers of structurally related furanoclerodanes. The biosynthetic capacity for formation of these diterpenoids is retained across most Lamiaceae subfamilies, including the early-diverging Callicarpoideae which forms a sister clade to the rest of Lamiaceae. VacCYP76BK1, a cytochrome P450 monooxygenase from Vitex agnus-castus, was previously found to catalyze the formation of the proposed precursor to furan and lactone-containing labdane diterpenoids. Through transcriptome-guided pathway exploration, we identified orthologs of VacCYP76BK1 in Ajuga reptans and Callicarpa americana. Functional characterization demonstrated that both could catalyze the oxidative cyclization of clerodane backbones to yield a furan ring. Subsequent investigation revealed a total of ten CYP76BK1 orthologs across six Lamiaceae subfamilies. Through analysis of available chromosome-scale genomes, we identified four CYP76BK1 members as syntelogs within a conserved syntenic block across divergent subfamilies. This suggests an evolutionary lineage that predates the speciation of the Lamiaceae. Functional characterization of the CYP76BK1 orthologs affirmed conservation of function, as all catalyzed furan ring formation. Additionally, some orthologs yielded two novel lactone ring moieties. The presence of the CYP76BK1 orthologs across Lamiaceae subfamilies closely overlaps with the distribution of reported furanoclerodanes. Together, the activities and distribution of the CYP76BK1 orthologs identified here support their central role in furanoclerodane biosynthesis within the Lamiaceae family. Our findings lay the groundwork for biotechnological applications to harness the economic potential of this promising class of compounds.

The Crystal Structure of Thermal Green Protein Q66E (TGP-E) and Yellow Thermostable Protein (YTP-E) E148D

Thermal green protein Q66E (TGP-E) has previously shown increased thermal stability compared to thermal green protein (TGP), a thermal stable fluorescent protein produced through consensus and surface protein engineering. In this paper, we describe the protein crystal structure of TGP-E to 2.0 Å. This structure reveals alterations in the hydrogen bond network near the chromophore that may result in the observed increase in thermal stability. We compare the very stable TGP-E protein to the structure of a yellow mutant version of this protein YTP-E E148D. The structure of this mutant protein reveals the rationale for the observed low quantum yield and directions for future protein engineering efforts.

Exploring ecotypic plant adaptations and the influence of microbiota on mitigating environmental challenges

The idea of ecotypes, which refer to unique groups of plants that have evolved to flourish in specific habitats, is gaining attention owing to climate fluctuations and changes in the associated microbiota. The term "ecotype" described plant populations that are specially adapted to particular environments, as revealed by common garden experiments showcasing genetically distinct characteristics. This concept remains relevant in modern plant science, where garden experiments continue to uncover how natural selection promotes species diversity, underscoring the crucial role of the plant microbiota in adaptation. Recent research has highlighted the microbial interactions that aid plants in adapting to environmental stress. Plants that shape soil microbial communities exhibit differential responses along ecological gradients owing to environmental stressors that influence interactions with soil microorganisms. Understanding the differentiation within plant populations and the emergence of new species is vital for discerning natural selection patterns. Environmental stressors profoundly impact global crop production, whereas plant-microbiota symbioses significantly influence plant growth and defense through nutrient acquisition and metabolite synthesis. Plant adaptation mechanisms include enzymatic antioxidant production and osmolyte accumulation, which are regulated by phytohormones that orchestrate responses to biotic and abiotic stressors, respectively. Despite breeding and genetic engineering efforts, progress in enhancing plant tolerance to extreme conditions remains limited, necessitating the development of sustainable agricultural alternatives. This study offers a comprehensive overview of recent advances in plant ecotype research, particularly focusing on symbiotic relationships with the microbiota and traits that contribute to improved nutrient uptake and plant health.

Bringing architecture-based adaption to the mainstream

Software architecture has been shown to provide an appropriate level of granularity for representation of a managed software system and reasoning about the impact of adaptation choices on its properties. Software architecture-based adaptability is the ability to adapt a software system in terms of its architectural elements, such as its components and their interfaces. Despite its promise, architecture-based adaptation has remained largely elusive, mainly because it involves heavy engineering effort of making non-trivial changes to the manner in which a software system is implemented. In this paper, we present Acadia—a framework that automatically enables architecture-based adaptation of practically any Java 9+ application without requiring any changes to the implementation of the application itself. Acadia builds on the Java Platform Module System (JPMS), which has brought extensive support for architecture-based development to Java 9 and subsequent versions. Acadia extends JPMS with the ability to provide and maintain a representation of an application’s architecture and make changes to it at runtime. The results of our experimental evaluation, conducted on three large open-source Java applications, indicate that Acadia is able to efficiently apply dynamic changes to the architecture of these applications without requiring any changes to their implementation.

Real-time low-light video enhancement on smartphones

Real-time low-light video enhancement on smartphones remains an open challenge due to hardware constraints such as limited sensor size and processing power. While night mode cameras have been introduced in smartphones to acquire high-quality images in light-constrained environments, their usability is restricted to static scenes as the camera must remain stationary for an extended period to leverage long exposure times or burst imaging techniques. Concurrently, significant process has been made in low-light enhancement on images coming out from the camera’s image signal processor (ISP), particularly through neural networks. These methods do not improve the image capture process itself; instead, they function as post-processing techniques to enhance the perceptual brightness and quality of captured imagery for display to human viewers. However, most neural networks are computationally intensive, making their mobile deployment either impractical or requiring considerable engineering efforts. This paper introduces VLight, a novel single-parameter low-light enhancement algorithm that enables real-time video enhancement on smartphones, along with real-time adaptation to changing lighting conditions and user-friendly fine-tuning. Operating as a custom brightness-booster on digital images, VLight provides real-time and device-agnostic enhancement directly on users’ devices. Notably, it delivers real-time low-light enhancement at up to 67 frames per second (FPS) for 4K videos locally on the smartphone.

Engineering and Characterization of a Long-Half-Life Relaxin Receptor RXFP1 Agonist.

Relaxin-2 is a peptide hormone with important roles in human cardiovascular and reproductive biology. Its ability to activate cellular responses such as vasodilation, angiogenesis, and anti-inflammatory and antifibrotic effects has led to significant interest in using relaxin-2 as a therapeutic for heart failure and several fibrotic conditions. However, recombinant relaxin-2 has a very short serum half-life, limiting its clinical applications. Here, we present protein engineering efforts targeting the relaxin-2 hormone in order to increase its serum half-life while maintaining its ability to activate the G protein-coupled receptor RXFP1. To achieve this, we optimized a fusion between relaxin-2 and an antibody Fc fragment, generating a version of the hormone with a circulating half-life of around 3 to 5 days in mice while retaining potent agonist activity at the RXFP1 receptor both in vitro and in vivo.

(Digital Presentation) Reactive Evaporation of Cr Vapors from Chromia-Alkaline Oxide Mixtures Generated in 800℃ Air and Subsequent Condensation Onto Aluminosilicate Fibers

This research aims to improve fundamental understanding of the reactive evaporation and condensation of Chromium (Cr) vapors, which are generated from Cr containing alloys used in many high-temperature (>500℃) process environments and can form potentially problematic condensed hexavalent (Cr(VI)) species downstream. This study focuses on the effects of alkaline oxides present in the Cr source on the resultant Cr evaporation and condensation. Vapor species were generated by flowing high-temperature (800℃) air containing 3% water vapor over chromia (Cr2O3) and mixtures of chromia with calcium oxide (CaO) or magnesium oxide (MgO) powders. Lab-grade quartz and alumina fiber samples were positioned downstream from the Cr-alkaline oxide source where the temperature decreases (<500℃) to collect condensed vapor species. Total condensed Cr and ratios of oxidation states were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES) and diphenyl carbazide (DPC) colorimetric/direct UV-VIS spectrophotometric analyses. Preliminary results indicate a decrease in partial pressures of generated Cr vapor species as well as a decrease in condensed Cr species on the aluminosilicate fibers when alkaline oxides are present in the Cr source compared to a pure Cr source. These results also indicate presence of hexavalent Cr (Cr(VI)) species condensed on all samples investigated. Computational thermodynamic equilibrium modelling corroborated the experimental results showing partial pressures of produced vapor species in the presence of alkaline oxides as well as stabilities of condensed Cr compounds on the fiber samples. Comparative results and analyses are presented and discussed to help inform mechanistic understanding and future related research and engineering efforts. Figure 1

Initial Value and Initial Current Discrepancy in Simulating Lithium-Ion Battery Packs - Resolution from Dr. Ralph White’s Analytical Approach

Battery models are useful tools that provide insight into electrochemical processes within an electrochemical device those can typically not be measured, either due to a lack of experimental methods or due to the transient nature of the phenomena in question. Irrespective of the complexity of the models involved, a general view is that a good model, once established for a single cell in terms of complexity of the physics, choice of simulation algorithm, value for the parameters, and mechanisms for fade, can be easily extended for pack simulations.1–3 This type of extension is critical to the virtual engineering efforts currently underway at OEMs which rely on accurate and representative battery models. In this work, pitfalls during scaling up approximate and detailed physics-based models developed for single-cell to pack-level simulations are highlighted using representative examples. Interesting mathematical nuances were found depending on the numerical simulation approach used. A discrepancy at initial times (t = 0) during the pack-level simulations was highlighted and resolved using the Laplace transform approach to get an analytical solution for the simplest model. A few thoughts on numerical challenges and the index of differential-algebraic equations (DAEs)4,5 while using the strong form and the weak form formulation of DAEs are also provided in the paper.6

In Vitro Encapsulation of Functionally Active Abiotic Photosensitizers Inside a Bacterial Microcompartment Shell.

Bacterial microcompartments (BMCs) are self-assembling, selectively permeable protein shells that encapsulate enzymes to enhance catalytic efficiency of segments of metabolic pathways through means of confinement. The modular nature of BMC shells' structure and assembly enables programming of shell permeability and underscores their promise in biotechnology engineering efforts for applications in industry, medicine, and clean energy. Realizing this potential requires methods for encapsulation of abiotic molecules, which have been developed here for the first time. We report in vitro cargo loading of BMC shells with ruthenium photosensitizers (RuPS) by two approaches─one involving site-specific covalent labeling and the other driven by diffusion, requiring no specific interactions between cargo molecules and shell proteins. The highly stable shells retain encapsulated cargo over 1 week without egress and preserve RuPS photophysical activity. This study is an important foundation for further work that will converge biological BMC architecture with synthetic chemistry to facilitate biohybrid photocatalysis.

KENYAMANAN TERMAL DI KAWASAN TENGAH KOTA JAKARTA

This research is to determine the level of thermal comfort based on occupant perception, microclimate and is associated with architectural engineering in human adaptation to urban heat island (UHI). One of the problems in cities is the increase in earth temperature caused by climate change and vertical obstruction. The impact of climate change and vertical obstruction will affect the level of human comfort in their environment. Thermal comfort is an absolute need for the human body, and with its ability, humans also create a built environment to fulfill their comfort both outside and inside a building. This research takes a case study at The Lavande Residence Apartment, which is a residence in the central area of ​​Jakarta. This research uses a descriptive analysis method from data obtained primarily through direct observation and interviews and several other secondary data. The research results show that the suitability of the comfort level based on climate simulations and the comfort calculator is at a neutral level and compared with the perception of residents who still feel comfortable. This perception of thermal comfort after being analyzed is due to architectural engineering efforts carried out by humans in the context of adaptation to climate change.

Genomic and Cell-Specific Regulation of Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy.

Opium poppy is a crop of great commercial value as a source of several opium alkaloids for the pharmaceutical industries including morphine, codeine, thebaine, noscapine and papaverine. Most enzymes involved in benzylisoquinoline alkaloids (BIAs) biosynthesis in opium poppy have been functionally characterized, and opium poppy currently serves as a model system to study BIA metabolism in plants. BIA biosynthesis in opium poppy involves two biosynthetic gene clusters associated respectively with the morphine and noscapine branches. Recent reports have shown that genes in the same cluster are co-expressed, suggesting they might also be co-regulated. However, the transcriptional regulation of opium poppy BIA biosynthesis is not well studied. Opium poppy BIA biosynthesis involves three cell types associated with the phloem system: companion cells, sieve elements and laticifers. The transcripts and enzymes associated with BIA biosynthesis are distributed across cell types, requiring the translocation of key enzymes and pathway intermediates between cell types. Together, these suggest that the regulation of BIA biosynthesis in opium poppy is multilayered and complex, involving biochemical, genomic, and physiological mechanisms. In this review, we highlight recent advances in genome sequencing and single cell and spatial transcriptomics with a focus on how these efforts can improve our understanding of the genomic and cell-specific regulation of BIA biosynthesis. Such knowledge is vital for opium poppy genetic improvement and metabolic engineering efforts targeting the modulation of alkaloid yield and composition.

Multi-omic characterization of antibody-producing CHO cell lines elucidates metabolic reprogramming and nutrient uptake bottlenecks

Characterizing the phenotypic diversity and metabolic capabilities of industrially relevant manufacturing cell lines is critical to bioprocess optimization and cell line development. Metabolic capabilities of production hosts limit nutrient and resource channeling into desired cellular processes and can have a profound impact on productivity. These limitations cannot be directly inferred from measured data such as spent media concentrations or transcriptomics. Here, we present an integrated multi-omic analysis pipeline combining exo-metabolomics, transcriptomics, and genome-scale metabolic network analysis and apply it to three antibody-producing Chinese Hamster Ovary cell lines to identify reprogramming features associated with high-producing clones and metabolic bottlenecks limiting product formation in an industrial bioprocess. Analysis of individual datatypes revealed a decreased nitrogenous byproduct secretion in high-producing clones and the topological changes in peripheral metabolic pathway expression associated with phase shifts. An integrated omics analysis in the context of the genome-scale metabolic model elucidated the differences in central metabolism and identified amino acid utilization bottlenecks limiting cell growth and antibody production that were not evident from exo-metabolomics or transcriptomics alone. Thus, we demonstrate the utility of a multi-omics characterization in providing an in-depth understanding of cellular metabolism, which is critical to efforts in cell engineering and bioprocess optimization.

Donor Sex and Passage Conditions Influence MSC Osteogenic Response in Mineralized Collagen Scaffolds.

Contemporary tissue engineering efforts often seek to use mesenchymal stem cells (MSCs) due to their multi-potent potential and ability to generate a pro-regenerative secretome. While many have reported the influence of matrix environment on MSC osteogenic response, few have investigated the effects of donor and sex. Here, a well-defined mineralized collagen scaffold is used to study the influence of passage number and donor-reported sex on MSC proliferation and osteogenic potential. A library of bone marrow and adipose tissue-derived stem cells from eight donors to examine donor viability in osteogenic capacity in mineralized collagen scaffolds is obtained. MSCs displayed reduced proliferative capacity as a function of passage duration. Further, MSCs showed significant sex-associated variability in osteogenic capacity. Notably, MSCs from male donors displayed significantly higher cell proliferation while MSCs from female donors displayed significantly higher osteogenic response via increased alkaline phosphate activity, osteoprotegerin release, and mineral formation in vitro. The study highlights the essentiality of including donor-reported sex as an experimental variable and reporting culture expansion in future studies of biomaterial regenerative potential.

Self-templated formation of ZnNC microtube with high oxygen reduction reaction overpotential for Zn-based catalyst

The synthesis of hollow morphologies without employing templates and addressing the high overpotential of the oxygen reduction reaction in Zn-N4-C-based materials poses a significant challenge. In this study, we present a straightforward yet robust approach based on a self-templated metal-organic framework (MOF-based) to synthesize ZnNC microtubes and explore the reason behind its high oxygen reduction (ORR) overpotential through density functional theory (DFT) calculation. ZnNC microtubes, benefiting from the abundant Zn-N4 active sites, hierarchical porosity, and a reasonable degree of graphitization, exhibited superior ORR activity and methanol tolerance compared to the traditional precious Pt/C catalyst. On the other hand, DFT calculation shows that Zn exhibits a d10 electronic configuration and diamagnetic nature in Zn-N4 moieties confirmed by the partial density of state (PDOS), hence the main reason behind the high ORR overpotential. This study provides a novel roadmap for further engineering efforts to achieve an ideal ORR catalyst in Zn-based materials.

Introducing carbon assimilation in yeasts using photosynthetic directed endosymbiosis

Conversion of heterotrophic organisms into partially or completely autotrophic organisms is primarily accomplished by extensive metabolic engineering and laboratory evolution efforts that channel CO2 into central carbon metabolism. Here, we develop a directed endosymbiosis approach to introduce carbon assimilation in budding yeasts. Particularly, we engineer carbon assimilating and sugar-secreting photosynthetic cyanobacterial endosymbionts within the yeast cells, which results in the generation of yeast/cyanobacteria chimeras that propagate under photosynthetic conditions in the presence of CO2 and in the absence of feedstock carbon sources like glucose or glycerol. We demonstrate that the yeast/cyanobacteria chimera can be engineered to biosynthesize natural products under the photosynthetic conditions. Additionally, we expand our directed endosymbiosis approach to standard laboratory strains of yeasts, which transforms them into photosynthetic yeast/cyanobacteria chimeras. We anticipate that our studies will have significant implications for sustainable biotechnology, synthetic biology, and experimentally studying the evolutionary adaptation of an additional organelle in yeast.

Preparation and characterization of poly(butylene adipate‐co‐terephthalate) composites reinforced with acetylated cellulose nanocrystals and nanofibers for enhanced properties

AbstractNanocellulose‐reinforced composites hold great promise for outperforming the original polymer matrix by offering exceptional properties, including outstanding barrier characteristics achieved through the establishment of convoluted pathways for the diffusion of water vapor and gases, as well as superior mechanical and thermal properties. Nevertheless, a significant challenge when employing nanocelluloses as reinforcements in polymers arises from their limited compatibility with the polymer matrix, primarily attributed to the abundance of surface hydroxyl groups.To address this limitation, this study focuses on the acetylation modification of cellulose nanocrystals (CNC) and nanofibers (CNF) to enhance their compatibility with the polymer matrix. Poly(butylene adipate‐co‐terephthalate) (PBAT) nanocomposites were developed by incorporating 1 wt% of both untreated and acetylated cellulose. The process involved the molten state mixing of these components using a twin‐screw mini extruder, conducted at 160°C for 7 min. The efficacy of the acetylation process was verified through Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) analysis confirmed the favorable interaction and dispersion of nanocelluloses within the polymer matrix. Importantly, introducing these fillers did not compromise thermal stability but notably enhanced the hydrophobic characteristics of the samples, as confirmed by contact angle measurements. Dynamic mechanical analyses further demonstrated the compatibility and effective interaction between the polymer and fillers. This research uniquely provides a comparative analysis of the effects of acetylated CNC and CNF within a PBAT matrix, unveiling distinct interactions and performance enhancements. Each type of nanocellulose exhibits its own characteristic ways of bonding and dispersing, leading to varying performance improvements for the material. This comparison elucidates the nuanced roles that these nanocelluloses play in reinforcing composites, offering insights into optimizing their contribution to polymer matrices. By demonstrating the differential impact of CNC and CNF post‐acetylation, our study contributes significantly to the field of polymer science, guiding future applications of nanocellulose‐reinforced materials in advanced engineering and sustainability efforts.