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mhryu@live.com
Today, 1:51 PM
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Conventional immunoassays such as ELISA are widely used in serological testing, yet their reliance on multi-step workflows and labeled reagents limits diagnostic scalability and speed. Bioluminescence-based biosensors are attractive alternatives that offer high sensitivity, operational simplicity, and cost efficiency for detecting diverse analytes. Here, we present a broadly compatible bioluminescent biosensor for antibody detection based on analyte-mediated reconstitution of split-Nanoluciferase (NanoLuc). The platform utilizes engineered bifunctional probes, comprising the protein G C2 domain fused to split-NanoLuc subunits (LgBiT and SmBiT), which serve dual functions in antibody binding and signal generation. Upon immunocomplex formation with multi-epitope antigens, the probes colocalize LgBiT and SmBiT, reconstituting NanoLuc activity and producing a quantifiable bioluminescent signal. We implemented this Fc-binding split-NanoLuc complementation assay to detect antibodies against African swine fever virus (ASFV). The optimized system showed high sensitivity, a wide linear dynamic range, and no cross-reactivity with sera positive for other common swine viruses. Clinical validation exhibited 97.11% agreement with a commercial ELISA kit, confirming its practicality and reliability. Furthermore, the sensor platform was seamlessly adapted to detect antibodies against SARS-CoV-2 and Chikungunya virus (CHIKV) without requiring additional molecular design or reconfiguration, highlighting its inherent versatility. By leveraging the broadly applicable Fc-binding capacity of protein G and the intrinsic modularity of split-NanoLuc, this strategy streamlines assay development and eliminates the need for species-specific secondary antibodies. Together, our findings demonstrate a proof-of-concept biosensor approach that could be further developed into a useful tool for rapid antibody detection in emerging infectious disease settings.
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mhryu@live.com
Today, 12:23 PM
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Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5′ side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved. Prokaryotic Class 2 CRISPR-Cas nucleases like Cas9 exhibit collateral cleavage activity. Here, the authors show that this activity is supported by unflanked R-loops in the RNA 5′ side, spacers of canonical length of 20 nt, and through its HNH and RuvC domains.
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mhryu@live.com
Today, 12:06 PM
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The gut microbiota communicates with multiple organs through neuroactive molecules, but the specific compounds produced remain unclear. We investigated eight commensal gut species, Lactococcus lactis, Enterococcus faecalis, Blautia producta, Clostridium symbiosum, Streptococcus thermophilus, Prevotella copri, Bacteroides fragilis, and Escherichia coli Nissle, using targeted and non-targeted LC-MS/MS. These bacteria differentially consumed glutamine, glutamate, and tryptophan, producing distinct neuroactive metabolites. For example, E. coli and B. producta generated high levels of gamma-aminobutyric acid (GABA), while P. copri was the sole producer of tryptamine. In the tyrosine pathway, E. faecalis synthesized tyramine and dopamine, and L. lactis, B. producta, and E. coli produced L-DOPA. Interestingly, none produced serotonin or its intermediates despite consuming tryptophan. Multiple species also generated short-chain fatty acids (SCFAs). These findings demonstrate that commensal microbes contribute to host neurochemistry by producing diverse neuroactive metabolites and SCFAs, highlighting the therapeutic potential of modulating the gut microbiota to influence human health.
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mhryu@live.com
Today, 11:36 AM
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The human reference proteome is routinely modeled with predictive tools such as AlphaFold2 and ESMFold. The two methods, based on different procedures, can behave differently depending on the experimental information available for a protein. We previously released a public database that stores pairs of predicted models, allowing us to obtain insights into the two methods and providing a resource where users can select the better model for downstream analysis. Here, we update the database after the latest release of UniProt (2025_04), we functionally characterize the models by mapping Pfam entries on the 3D structures, and we introduce external quality assessment metrics to evaluate and compare the models. We observe that, regardless of the quality and similarity of the predicted models, both AlphaFold2 and ESMFold converge with high pLDDT values in regions covered by Pfam entries. Alpha&ESMhFolds, including all its features, is freely available at https://alpha-esmhfolds.biocomp.unibo.it/.
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mhryu@live.com
Today, 10:43 AM
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Adenine deaminase (ADE) plays a critical role in food purine degradation and in preventing hyperuricemia. Herein, the ADE genes from four different sources were expressed using pMA5, pMA0911, and pP43NMK as the vectors and Bacillus subtilis WB800 as the host. B. subtilis WB800/p43-AK-ADE exhibited the highest ADE activity of 45.41 U/mL. The optimum pH and temperature for AK-ADE were 7.5 and 45 °C, respectively. The enzyme remained stable within pH 6.0–8.5 and temperature 35–45 °C, respectively. The Km and Kcat values were 0.30 mM and 0.28 s–1, respectively. Furthermore, molecular docking and active pocket prediction revealed that AK-ADE binds to adenine with the lowest binding free energy, indicating the strongest affinity. In contrast, ADE from other sources exhibited markedly reduced binding and suboptimal residue positioning. Finally, when AK-ADE was applied to beer wort, it resulted in the removal of 71% of the adenine content, establishing its potential for industrial application.
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mhryu@live.com
January 24, 7:58 PM
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This special issue marking the University of Bath's 60th anniversary offers an opportunity to reflect on nearly a decade of research into the evolution of gene regulatory networks (GRNs) from members of the lab and elsewhere. Our goal is to understand how GRNs rewire and how new transcription factor (TF) functions evolve. Using an experimental evolution model system with the soil bacterium Pseudomonas fluorescens, we have been able to observe TF rewiring in real time, providing unique insights into the principles of GRN evolution. In this perspective, we highlight three central discoveries from this system: a hierarchical pattern of TF rewiring, in which some regulators act as preferred “first responders”; the critical influence of expression level and mutational accessibility on whether a TF can be recruited for novel function; and the role of crosstalk (non-cognate binding) as the raw material for adaptive innovation. Together, these findings reveal why evolutionary pathways are often constrained and thus strikingly repeatable. By identifying what makes a TF evolvable, we are beginning to predict, and potentially direct, evolutionary outcomes. Finally, we consider open questions and emerging technologies that have the potential to transform our understanding of GRN rewiring and its relationship with evolvability.
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mhryu@live.com
January 24, 7:47 PM
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Bacterial cellulose (BC) hydrogels produced by Gluconacetobacter species hold considerable promise for a wide range of applications owing to their exceptional mechanical properties, biocompatibility, and biodegradability. Achieving precise control over their structural and mechanical characteristics is crucial for the engineering of BC-based materials. In this study, we investigated the formation dynamics and structural features of BC hydrogels, emphasizing the complex interplay between cellulose nanofibril secretion and bacterial motility. Comprehensive tracking of bacterial movement during hydrogel formation has validated mechanisms underlying the development of branching and merging junctions, which are key elements that define the network's physical properties. Additionally, we observed the emergence of vortex-lattice and chiral-nematic structures during hydrogel development, depending on bacterial and cellulose densities. These insights contribute to a fundamental understanding of bottom-up 3D fabrication of BC hydrogels that harness the collective behavior of cellulose-producing bacteria.
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mhryu@live.com
January 24, 7:37 PM
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mRNA medicines hold great promise, but designing sequences with high translation efficiency, robust in-solution stability, and manufacturability remains a major challenge due to the vast combinatorial space of synonymous coding sequences. Computational approaches such as mRNA folding algorithms have emerged as powerful tools by co-optimizing for in-solution stability and translation efficiency, yet current methods face important limitations. Here, we present "mRNAfold", an improved mRNA folding algorithm and software package that addresses these gaps by enabling efficient exploration of diverse near-optimal solutions, incorporating untranslated regions (UTRs), parallel execution, and supporting tunable control over local structural features across the mRNA. Thermodynamically optimized mRNAs from mRNAfold were more stable (≈2-fold) in-solution than those generated by simple GC maximization for the same encoded protein. In addition, mRNAs designed to vary local structure near the start codon while maintaining consistent structure and codon optimality elsewhere showed a complex relationship between local structure near the start codon and protein production in cells. We observed no impact of structure in the start codon region for a set of mRNAs with high codon optimality, but it did impact protein production for a set of mRNAs with lower codon optimality. Together, these results underscore the potential of structure-aware, multi-objective design to improve mRNA medicines and offer a framework for exploring how sequence, structure, and expression are interrelated.
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mhryu@live.com
January 24, 7:24 PM
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Transposon insertion sequencing (TIS) encompasses methods such as Transposon Directed Insertion sequencing (TraDIS) and Transposon-Sequencing (Tn-Seq); these methods are widely uses for genome scale screens of essential and conditionally essential genes. Early limitations centred on mutant generation, today the biggest factor is sequencing and data generation and the costs associated. Here, we present and evaluate four methods TIS sequencing library generation protocols compatible with Illumina whole genome sequencing (WGS) workflows to maximise sequence efficiency and minimise turnaround times and costs. Using E.coli BW25113 transposon mutagenesis libraries generated with Tn5 and mariner (Himar1) transposons, we compare the methods in terms of reagent cost, workflow complexity, and target enrichments for the recovery of unique insertion sites and essential genes identified. All methods generated TIS data suitable for gene essentiality analysis. Illumina Flex based protocols were 4-6 times cheaper than the traditional Illumina Nextera based approach with similar or superior Transposon-Chromosome (Tn-Chr) junction enrichment. Across all methods, sequencing depth and mutant library density were the dominant factor in useful biological insight. Subsampling demonstrated that for a good quality mutant library, five million reads were sufficient to identify essential genes in E.coli. Whereas deeper sequencing reduced the statistical power and included contaminating background noise, seen primarily with Tn5. We conclude that an Illumina Flex based approach, especially when integrating with routine WGS, provides an excellent balance of speed, cost and data quality. Assuming five million reads and a robust Illumina Flex approach, a TIS library can be sequenced for around £40.
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mhryu@live.com
January 23, 5:29 PM
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To counter challenges from bacteriophages (phages), bacteria use defense mechanisms that can reside on mobile genetic elements or within chromosomes. These immune systems are easily gained and lost, allowing adaptation to threats. However, the mechanism of mobilization of chromosomally encoded defense genes remains poorly understood. Here, we show that phage- and phage-inducible chromosomal island (PICI)–mediated lateral transduction (LT), a highly efficient horizontal gene transfer HGT mechanism, facilitates the transfer of these defense genes between bacteria. Using several bacterial models, we demonstrate that defense systems are often positioned near phage or PICI attachment sites, allowing them to exploit LT for their mobility. In addition, LT diversifies defense genes carried by prophages and PICIs, driving immune system evolution and turnover. These processes provide phage resistance to new bacterial hosts and profoundly affect population genomics. Our findings reveal LT as a crucial mechanism shaping bacterial evolution and influencing the trajectory of pathogenic clones in nature.
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mhryu@live.com
January 23, 5:24 PM
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Terpenoid-based biofuels represent a sustainable alternative to fossil fuels with superior energy densities and combustion properties. However, achieving industrial-scale production requires overcoming multiple bottlenecks: heterologous enzyme incompatibility, metabolic flux imbalance, product toxicity, and economic viability. This review synthesizes recent breakthroughs in addressing these challenges through enzyme engineering, metabolic rewiring, host tolerance enhancement, and feedstock utilization. Simultaneously, the field is positioned at a critical juncture where convergent technologies — generative artificial intelligence for protein discovery, synthetic organelles via liquid–liquid phase separation LLPS, and engineering of non-natural terpenoid scaffolds (C₁₁, C₁₆) — promise transformative advances. This review provides a roadmap integrating these emerging capabilities to advance terpenoid-based biofuels toward commercial viability.
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mhryu@live.com
January 23, 5:15 PM
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Xanthan gum, a natural heteropolysaccharide produced by Xanthomonas species, has many biotechnological applications across industries due to its unique rheological properties. Expanding its utility requires specific enzymes capable of targeted xanthan modification or degradation. In this study, a novel bacterial strain, isolated from a spoiled xanthan sample and identified as Paenibacillus taichungensis I5, was shown to degrade xanthan using a plate screening assay with Congo red. Activity tests of crude enzyme in culture supernatant demonstrated the secretion of xanthan-degrading enzymes. Genome and proteome analyses suggest a chromosomal xanthan utilization locus encoding a suite of enzymes, including a xanthanase (Pt_XanGH9), two xanthan lyases (Pt_XanPL8a and Pt_XanPL8b), two unsaturated glucuronidases, two α-mannosidases, as well as transport and regulator proteins. Functional characterization through recombinant protein expression and enzyme assays confirmed the functions of Pt_XanGH9, Pt_XanPL8a and Pt_XanPL8b on native xanthan and xanthan-derived oligosaccharides. The polysaccharide degradation products released by these enzymes were identified via LC–MS analysis and suggested two xanthan lyases with divergent cleavage preferences. In contrast to Pt_XanPL8a, Pt_XanPL8b is synthesized with an N-terminal signal peptide, yet both lyases were detected in cell-free supernatant during growth on xanthan. Based on the composition of the xanthan utilization gene cluster and preliminary enzyme characteristics, a working model for xanthan utilization by P. taichungensis I5 is proposed. Reaching a better understanding of bacterial xanthan degrading pathways and the enzymes involved may help to develop modified xanthan derivatives and xanthan degrading enzymes that align with the specific demands of various industrial process.
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mhryu@live.com
January 23, 5:09 PM
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In natural and engineered ecosystems, diverse species interact in complex ways to form highly efficient microecologies. One key orchestrator of these interactions is autoinducer-2 (AI-2), a signaling molecule that plays a crucial role in microbial community assembly, metabolic flux, and resilience to environmental disturbances. This review provides the systematic synthesis of AI-2’s dual structural dynamics (S-THMF-borate/R-THMF interconversion) and its context-dependent roles in mediating bacterial crosstalk. It also reveals the receptor diversity (such as LuxP and LsrB) of AI-2 in bacterial kingdom and the signal transduction mechanism. Systematically elaborated on AI-2’s regulation of cellular metabolic flux and its ability to autonomously exhibit a series of coordinated behaviors in response to environmental changes. The review explores the ramifications of AI-2 on bacterial community interactions in synthetic biology and natural ecosystems. The wide application of AI-2-mediated interspecific communication in various fields including host health, agriculture, industry and environmental ecology has also been widely discussed. Factors influencing AI-2 production are thoroughly examined, including internal factors such as strain specificity, cell density, growth form and the phenotypic heterogeneity. Additionally, external biological factors (such as nutritional status and environmental stress) and abiotic factors (aggregation, diffusion, and flow) are discussed in detail. By examining knowledge gaps in AI-2-mediated spatial heterogeneity and multi-QS system coordination, this work charts a roadmap for harnessing microbial communication in chemical engineering and environmental sustainability.
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mhryu@live.com
Today, 1:29 PM
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Microbial transglutaminase (mTG) is an enzyme produced by actinomycetes, predominantly by filamentous bacteria belonging to the genus Streptomyces. These microorganisms are the best-studied and most microbiologically exploited natural producers of TG. It catalyzes the formation of isopeptide bonds between glutamine and lysine residues in proteins, which alters the protein’s structure and functionality. This enzymatic activity is widely applied in the food industry, where it is particularly useful in modifying the texture, binding properties, and overall quality of protein products. The primary application of mTG in food production lies in its ability to mimic the functional properties of gluten, making it valuable in the creation of gluten-free products. By facilitating the binding of various ingredients such as starches and plant proteins, mTG helps produce products with similar texture and elasticity to those made with gluten, which is crucial for individuals with celiac disease or gluten intolerance. Beyond gluten-free applications, mTG is also employed in the production of meat substitutes, where it enhances the texture and cohesion of plant-based ingredients, as well as in bakery and confectionery products. Additionally, mTG is utilized in the development of organic materials, such as microcapsules and enzymatic carriers, owing to its calcium-independent activity, broad substrate specificity, and high stability across a wide range of pH and temperatures. The properties of this enzyme can be successfully used in the biotechnology industry. Looking forward, mTG is expected to play a significant role in the expansion of functional foods, plant-based diets, and advanced biomaterials. However, there are growing concerns about its safety, particularly regarding potential immunogenic reactions and its long-term impact on consumer health. To mitigate these risks, further research on the biological effects of mTG is essential, especially in the context of its microbial origin, molecular structure, and interaction with human proteins. Additionally, stricter regulations and clear labeling of products containing mTG will be necessary to ensure consumer safety and confidence in its widespread use.
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mhryu@live.com
Today, 12:10 PM
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Microbial metabolic gene clusters encode the biosynthesis or catabolism of metabolites that facilitate ecological specialization, mediate microbiome interactions and constitute a major source of medicines and crop protection agents. Here, we present BiG-SCAPE and BiG-SLiCE 2.0, next-generation methods that facilitate scalable, accurate and interactive gene cluster analyses. BiG-SCAPE 2.0 updates its classification, alignment methods, and visualizations, enabling more accurate analysis, up to 8x faster runtimes and halved memory requirements. BiG-SLiCE 2.0 updates its distance metric, pHMM database, and classification logic, resulting in increased sensitivity nearing that of BiG-SCAPE. Analysis of 260,630 biosynthetic gene clusters from publicly available genomes reveals that both tools generate concurring estimates of gene cluster diversity, thus providing significantly extended methodological support for recent evidence indicating that the vast majority of natural product diversity remains unexplored. Together, these updates will facilitate global genome mining efforts for natural product discovery and microbiome analyses scalable with current data sizes. BiG-SCAPE and BiG-SLiCE are computational tools that enable exploring the diversity of metabolic gene clusters across microbial genomes. Here, the authors present major updates to these tools, providing essential infrastructure for studying the diversity of microbial metabolism.
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mhryu@live.com
Today, 11:52 AM
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Deep computationally guided protein design enabled the introduction of a Ser-His-Asp catalytic triad that supports polyethylene terephthalate (PET) hydrolysis within an inner membrane E. coli protein. This allows the engineering, through gene editing, of a strain capable of degrading PET particles smaller than 4.5 nm. We extended this approach to PET powder (<300 μm) using a computational workflow that builds geometrically pre-organized catalytic triads while preserving substrate binding in extracellular or surface-exposed membrane proteins. Four additional proteins were reprogrammed to degrade PET. Replacement of the outer membrane protein OmpA with a PETase-active variant carrying a surface-exposed artificial triad enabled an engineered strain to release 157 ± 2 μM hydrolysis products within 24 h at 37°C and to sustain growth (0.18 ± 0.07 h−1) on PET powder as a carbon source. These results demonstrate the feasibility of engineering E. coli for PET powder biodegradation without exogenous PETase genes.
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mhryu@live.com
Today, 10:50 AM
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Global warming driven by carbon emissions makes transitioning to a circular carbon economy increasingly urgent. Methanol is a promising energy carrier and a favorable substrate for biotechnology applications due to its high energy conversion efficiency and easy integration with existing infrastructure. Eubacterium limosum has the native ability to convert methanol into valuable chemicals, including acetate, butyrate, and hexanoate. However, oxidized cosubstrates such as CO2 or formate are generally required due to limitations in energy and redox balance. In this study, we demonstrate a novel approach using extracellular electron transfer (EET) with ferric iron (ferrihydrite) as an electron acceptor to enable methanol fermentation without carbon cosubstrates. This ferrihydrite-assisted methanol fermentation yielded a product spectrum like methanol-formate cofermentation, producing more reduced chemicals such as hexanoate. Physiological evidence indicated that cysteine (Cys-SH), an amino acid containing a thiol group (R-SH) and typically included in anaerobic media, was essential in enabling the indirect EET process between E. limosum and ferrihydrite. Thiol regeneration from cystine (Cys–S–S–Cys) confirmed that E. limosum catalyzed the cysteine/cystine recycling. Proteomic analysis revealed the metabolic pathways of this cysteine-mediated EET process. This EET-driven process advances our understanding of microbial carbon cycling and provides a sustainable biotechnology for chemical production.
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mhryu@live.com
Today, 10:38 AM
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Wearable biosensors are an emerging field in the area of health monitoring, and when they combine with various biopolymers, they provide affordable, sustainable, noninvasive, and real-time monitoring of health. Natural biopolymers such as silk, cellulose, and chitosan offer great potential in the fabrication of these biosensors because of their various properties, such as biocompatibility, flexibility, biodegradability, hydrophilicity, and renewability. These biopolymer-based wearable biosensors provide continuity and comfort with high precision in monitoring health in less time. This Account explores how the remarkable structural and physicochemical properties of these biopolymers support their fabrication and integration into wearable biosensors that can withstand the dynamic environment of the human body. Leveraging these biopolymers enables the development of eco-friendly and skin-conformable biosensors for glucose, lactate, and other biofluids including saliva, sweat, tears, and other interstitial fluids. These biopolymers are of significant importance in the domains of personalized medicine, enhancing athletic performance tracking, and chronic disease management for next-generation wearable devices.
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mhryu@live.com
January 24, 7:52 PM
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Heme is an essential cofactor and dietary source of iron for the obligate human pathogen, Mycobacterium tuberculosis (Mtb). Consequently, heme is required for Mtb growth and pathogenicity and strategies to limit heme represents a promising therapeutic approach. Although Mtb can both make and scavenge heme, it was previously found that de novo synthesized heme is substantially more bioavailable and metabolically active than exogenously scavenged heme. These findings provided a strong justification to target the terminal heme biosynthetic enzyme, coproheme decarboxylase (ChdC), in the development of anti-mycobacterial therapies. Herein, we sought to characterize heme homeostasis in a ΔchdC deletion mutant in Mtb. Surprisingly, we found that ablation of ChdC in Mtb and Mycobacterium smegmatis (Msm) resulted in the enhanced accumulation and bioavailability of exogenously scavenged heme compared to wild type or mutants lacking glutamyl tRNA reductase (GtrR), the first enzyme in the heme synthetic pathway. Moreover, we found that Mtb has a preference for scavenging reduced ferrous heme and exhibits a heme reductase activity that is inhibited by ChdC. We further found that ChdC expression is down-regulated when iron is limiting, which in-turn increases both heme import and bioavailability. Such a mechanism may serve to protect cells from heme toxicity while trying to meet the nutritional demand for iron. Importantly, our results also suggest caution must be taken if targeting ChdC due to feedback mechanisms that lead to enhanced heme scavenging in response to ChdC ablation.
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mhryu@live.com
January 24, 7:41 PM
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Concrete is an extensively used construction material in infrastructures due to cost-effectiveness and durability. However, its low tensile strength makes it prone to cracking, posing persistent maintenance challenges. Traditional repair methods are often expensive and difficult to implement, especially for critical structures. Recent studies propose a potential solution involving alkaliphilic or alkali-tolerant calcium-carbonate producing bacteria in aiding concrete crack repair through microbial-induced calcium carbonate precipitation (MICP) via urea hydrolysis. Considering the adaptive nature of bacteria, we hypothesize that naturally occurring concrete-inhabiting microbes possess properties crucial for mending cracks. Therefore, this study aims to isolate and evaluate these inherent microbes and their MICP ability. Six concrete samples were collected and cultured on Alkaline Nutrient Agar for microbial isolation. Urease activity was assessed phenotypically and genotypically. MICP activity was observed and validated under Scanning Electron Microscope/Energy Dispersive Spectroscopy (SEM/EDS). Out of the 49 isolates cultured, Lysinibacillus sphaericus PUMA0250 emerges as the most promising isolate for practical implementation, showing high pH survivability in an in-house formulated concrete agar medium (pH 12.6). L. sphaericus PUMA0250 exhibited MICP activity via urea hydrolysis and harbored all crucial genes involved in urease-mediated MICP. SEM/EDS analysis confirmed the structure and composition of the produced calcium carbonate precipitate. This study demonstrates the potential of L. sphaericus PUMA0250 to be incorporated into concrete to assess its healing efficiency. Its ability to thrive in the alkaline concrete environment and produce calcium by-products may aid concrete crack repair, opening microbiological avenues for sustainable and efficient concrete repair methodologies.
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mhryu@live.com
January 24, 7:30 PM
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Polyethylene terephthalate (PET) is one of the most widely used plastics and a major contributor to marine pollution. While the diversity of PET hydrolases (PETases), which degrade PET into mono(2-hydroxyethyl) terephthalate (MHET), terephthalate (TPA) and ethylene glycol, has been documented in temperate and tropical waters, their potential presence in polar oceans remain unascertained. Here, we systematically screened polar and non-polar marine metagenomes using Hidden Markov models (HMM) generated using experimentally validated PETases. We identified >680 putative PETase-like sequences, with Antarctic and Arctic candidates enriched in high-fidelity motifs associated with PETase-like activity. Phylogenetic and structural analyses defined a high-confidence PETase-like clade comprising both Type I and Type II enzymes, differing in thermostability-related features and PET-binding motifs. Experimental assays confirmed polyesterase activity in 5/9 candidates from this clade, including polar-derived variants active at 14-25°C. Downstream enzymes for PET consumption were also widespread, detecting 209 putative MHET hydrolases and 442 TPA-catabolyzing enzymes. Further, we reconstructed 112 metagenome-assembled genomes (MAGs) carrying at least one PETase-like gene, more than half from polar datasets. Notably, 15 MAGs encoded multiple PETase-like enzymes, and 1 Antarctic MAG harbored a complete PETase-MHETase-TPA pathway, evidencing a fully integrated degradation potential in cold-adapted taxa. Together, these results demonstrate that polar oceans act as previously overlooked reservoirs of taxonomically and functionally diverse plastic-degrading enzymes. The enrichment of PETase-like enzymes and downstream pathways in polar microbial communities expands the global biogeography of plastic biodegradation and highlights cold-active enzymes as promising candidates for developing low-temperature plastic bioremediation strategies.
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mhryu@live.com
January 24, 7:20 PM
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Eukaryotic gene expression is a multi-layered process influenced by multiple factors. One of them is the secondary structure of precursor mRNAs that can impact various aspects of their processing including alternative splicing (AS). Here, we report the functional characterization of the conserved RNA structural element DEAD that is located in DEAD-box RNA helicase (DRH) genes from land plants and serves as a sensor for RNA helicase activity by controlling AS. In Arabidopsis thaliana, it is found in DRH1 and its closest paralog, regulating usage of an alternative splice site as part of a negative feedback loop. Accordingly, opening of the structure shifts splicing towards non-coding variants, thereby balancing transcript and protein levels. Interestingly, the system is specific to DRH1 and its paralog and does not react to related helicases, which is at least partially conferred by the disordered and RGG/RG motif-containing C-terminus of DRH1. The importance of DEAD is underlined by the observation that releasing this attenuation mechanism causes massive changes in AS - mainly intron retention and exon skipping - and gene expression and results in a severe stress phenotype. Thus, DEAD provides a critical buffering mechanism to fine-tune helicase levels and their global impact on RNA structure-responsive gene expression.
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mhryu@live.com
January 23, 5:26 PM
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Steroid hormones are key signaling molecules regulating growth, metabolism, reproduction, and stress adaptation and are widely used as essential pharmaceuticals. Traditional production from sterol feedstocks through multistep chemical or microbial transformations is limited by inefficiency and scalability. Recent advances in synthetic biotechnology enable de novo biosynthesis of steroid hormones from simple carbon sources in yeasts and fungi. This review highlights metabolic rewiring to increase flux, cytochrome P450 enzyme engineering for side-chain cleavage, and hydroxylation to overcome rate-limiting bottlenecks of steroid hormone biosynthesis. We also discuss strategies to redesign steroid-transport pathways to alleviate intracellular accumulation and improve membrane export. Looking ahead, we envision integrating metabolic, enzyme, and transport engineering to build a scalable, data-driven ‘intelligent’ platform for sustainable steroid hormone biomanufacturing.
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mhryu@live.com
January 23, 5:17 PM
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Development of chemically modified oligonucleotides, nucleic acid mimics, protein-based constructs, and other ligands -capable of sequence-unrestricted recognition of specific double-stranded (ds) DNA regions -is an area of research that continues to attract considerable attention. Efforts are fueled by the need for diagnostic agents, modulators of gene expression, and novel therapeutic modalities against genetic diseases. While pioneering approaches focused on accessing nucleotide-specific features from the grooves of DNA duplexes, recent developments have entailed strand-invading probes, i.e., probes capable of binding to DNA duplexes by breaking existing Watson-Crick base pairs and forming new, more stable base pairs. For the past twenty years, our laboratory has pursued the development of a type of dsDNA-targeting strand-invading probes, which we have named Invader probes. These double-stranded oligonucleotide probes feature intercalator-functionalized nucleotides that are specifically arranged to promote destabilization of the probe duplex, whereas individual strands exhibit very high affinity towards complementary DNA. This account details the discovery, principles, and applications of Invader probes.
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mhryu@live.com
January 23, 5:13 PM
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Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing. We recently established a transgene-free VIGE approach for heritable editing at single loci in Arabidopsis by delivering ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV). Here, we greatly improved this approach by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) to develop an efficient multiplexed genome editing approach.
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gaut, A 3D homology model of AK-ADE was built using SWISS-MODEL (http://www.swissmodel.expasy.org) and validated via the SAVES 6.1 server (http://saves.mbi.ucla.edu/). Potential active pockets were predicted using DoGSiteScorer (https://proteins.plus/). The protein structure was prepared for docking by removing water molecules and ligands using the PyMOL software. The processed protein was imported into AutoDock Vina and set as the receptor, while the substrate adenine was imported and selected as the ligand.