Insights into the Main Protease of SARS-CoV-2: Thermodynamic Analysis, Structural Characterization, and the Impact of Inhibitors.

The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for coronaviral maturation and is the target of Paxlovid, which is currently the standard-of-care treatment for COVID-19. There remains a need to identify new inhibitors of Mpro as viral resistance to Paxlovid emerges. Here, we report the use of native mass spectrometry coupled with 193 nm ultraviolet photodissociation (UVPD) and integrated with other biophysical tools to structurally characterize Mpro and its interactions with potential covalent inhibitors. The overall energy landscape was obtained using variable temperature nanoelectrospray ionization (vT-nESI), thus providing quantitative evaluation of inhibitor binding on the stability of Mpro. Thermodynamic parameters extracted from van't Hoff plots revealed that the dimeric complexes containing each inhibitor showed enhanced stability through increased melting temperatures as well as overall lower average charge states, giving insight into the basis for inhibition mechanisms.

Serial adaptive laboratory evolution enhances mixed carbon metabolic capacity of Escherichia coli.

Microbes have inherent capacities for utilizing various carbon sources, however they often exhibit sub-par fitness due to low metabolic efficiency. To test whether a bacterial strain can optimally utilize multiple carbon sources, Escherichia coli was serially evolved in L-lactate and glycerol. This yielded two end-point strains that evolved first in L-lactate then in glycerol, and vice versa. The end-point strains displayed a universal growth advantage on single and a mixture of adaptive carbon sources, enabled by a concerted action of carbon source-specialists and generalist mutants. The combination of just four variants of glpK, ppsA, ydcI, and rph-pyrE, accounted for more than 80% of end-point strain fitness. In addition, machine learning analysis revealed a coordinated activity of transcriptional regulators imparting condition-specific regulation of gene expression. The effectiveness of the serial adaptive laboratory evolution (ALE) scheme in bioproduction applications was assessed under single and mixed-carbon culture conditions, in which serial ALE strain exhibited superior productivity of acetoin compared to ancestral strains. Together, systems-level analysis elucidated the molecular basis of serial evolution, which hold potential utility in bioproduction applications.

Synthetic 3'-UTR valves for optimal metabolic flux control in Escherichia coli.

As the design of genetic circuitry for synthetic biology becomes more sophisticated, diverse regulatory bioparts are required. Despite their importance, well-characterized 3'-untranslated region (3'-UTR) bioparts are limited. Thus, transcript 3'-ends require further investigation to understand the underlying regulatory role and applications of the 3'-UTR. Here, we revisited the use of Term-Seq in the Escherichia coli strain K-12 MG1655 to enhance our understanding of 3'-UTR regulatory functions and to provide a diverse collection of tunable 3'-UTR bioparts with a wide termination strength range. Comprehensive analysis of 1,629 transcript 3'-end positions revealed multiple 3'-termini classes generated through transcription termination and RNA processing. The examination of individual Rho-independent terminators revealed a reduction in downstream gene expression over a wide range, which led to the design of novel synthetic metabolic valves that control metabolic fluxes in branched pathways. These synthetic metabolic valves determine the optimal balance of heterologous pathways for maximum target biochemical productivity. The regulatory strategy using 3'-UTR bioparts is advantageous over promoter- or 5'-UTR-based transcriptional control as it modulates gene expression at transcription levels without trans-acting element requirements (e.g. transcription factors). Our results provide a foundational platform for 3'-UTR engineering in synthetic biology applications.

Graph Neural Network Model for Prediction of Non-Small Cell Lung Cancer Lymph Node Metastasis Using Protein-Protein Interaction Network and 18F-FDG PET/CT Radiomics.

The image texture features obtained from 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) images of non-small cell lung cancer (NSCLC) have revealed tumor heterogeneity. A combination of genomic data and radiomics may improve the prediction of tumor prognosis. This study aimed to predict NSCLC metastasis using a graph neural network (GNN) obtained by combining a protein-protein interaction (PPI) network based on gene expression data and image texture features. 18F-FDG PET/CT images and RNA sequencing data of 93 patients with NSCLC were acquired from The Cancer Imaging Archive. Image texture features were extracted from 18F-FDG PET/CT images and area under the curve receiver operating characteristic curve (AUC) of each image feature was calculated. Weighted gene co-expression network analysis (WGCNA) was used to construct gene modules, followed by functional enrichment analysis and identification of differentially expressed genes. The PPI of each gene module and genes belonging to metastasis-related processes were converted via a graph attention network. Images and genomic features were concatenated. The GNN model using PPI modules from WGCNA and metastasis-related functions combined with image texture features was evaluated quantitatively. Fifty-five image texture features were extracted from 18F-FDG PET/CT, and radiomic features were selected based on AUC (n = 10). Eighty-six gene modules were clustered by WGCNA. Genes (n = 19) enriched in the metastasis-related pathways were filtered using DEG analysis. The accuracy of the PPI network, derived from WGCNA modules and metastasis-related genes, improved from 0.4795 to 0.5830 (p < 2.75 × 10-12). Integrating PPI of four metastasis-related genes with 18F-FDG PET/CT image features in a GNN model elevated its accuracy over a without image feature model to 0.8545 (95% CI = 0.8401-0.8689, p-value < 0.02). This model demonstrated significant enhancement compared to the model using PPI and 18F-FDG PET/CT derived from WGCNA (p-value < 0.02), underscoring the critical role of metastasis-related genes in prediction model. The enhanced predictive capability of the lymph node metastasis prediction GNN model for NSCLC, achieved through the integration of comprehensive image features with genomic data, demonstrates promise for clinical implementation.

Adaptive laboratory evolution of Escherichia coli W enhances gamma-aminobutyric acid production using glycerol as the carbon source.

The microbial conversion of glycerol into value-added commodity products has emerged as an attractive means to meet the demands of biosustainability. However, glycerol is a non-preferential carbon source for productive fermentation because of its low energy density. We employed evolutionary and metabolic engineering in tandem to construct an Escherichia coli strain with improved GABA production using glycerol as the feedstock carbon. Adaptive evolution of E. coli W under glycerol-limited conditions for 1300 generations harnessed an adapted strain with a metabolic system optimized for glycerol utilization. Mutation profiling, enzyme kinetic assays, and transcriptome analysis of the adapted strain allowed us to decipher the basis of glycerol adaptation at the molecular level. Importantly, increased substrate influx mediated by the mutant glpK and modulation of intracellular cAMP levels were the key drivers of improved fitness in the glycerol-limited condition. Leveraging the enhanced capability of glycerol utilization in the strain, we constructed a GABA-producing E. coli W-derivative with superior GABA production compared to the wild-type. Furthermore, rationally designed inactivation of the non-essential metabolic genes, including ackA, mgsA, and gabT, in the glycerol-adapted strain improved the final GABA titer and specific productivity by 3.9- and 4.3-fold, respectively, compared with the wild-type.

Engineering Bacteroides thetaiotaomicron to produce non-native butyrate based on a genome-scale metabolic model-guided design.

Bacteroides thetaiotaomicron represents a major symbiont of the human gut microbiome that is increasingly viewed as a promising candidate strain for microbial therapeutics. Here, we engineer B. thetaiotaomicron for heterologous production of non-native butyrate as a proof-of-concept biochemical at therapeutically relevant concentrations. Since B. thetaiotaomicron is not a natural producer of butyrate, we heterologously expressed a butyrate biosynthetic pathway in the strain, which led to the production of butyrate at the final concentration of 12 mg/L in a rich medium. Further optimization of butyrate production was achieved by a round of metabolic engineering guided by an expanded genome-scale metabolic model (GEM) of B. thetaiotaomicron. The in silico knock-out simulation of the expanded model showed that pta and ldhD were the potent knock-out targets to enhance butyrate production. The maximum titer and specific productivity of butyrate in the pta-ldhD double knockout mutant increased by nearly 3.4 and 4.8 folds, respectively. To our knowledge, this is the first engineering attempt that enabled butyrate production from a non-butyrate producing commensal B. thetaiotaomicron. The study also highlights that B. thetaiotaomicron can serve as an effective strain for live microbial therapeutics in human.

System-level understanding of gene expression and regulation for engineering secondary metabolite production in Streptomyces.

The gram-positive bacterium, Streptomyces, is noticed for its ability to produce a wide array of pharmaceutically active compounds through secondary metabolism. To discover novel bioactive secondary metabolites and increase the production, Streptomyces species have been extensively studied for the past decades. Among the cellular components, RNA molecules play important roles as the messengers for gene expression and diverse regulations taking place at the RNA level. Thus, the analysis of RNA-level regulation is critical to understanding the regulation of Streptomyces' metabolism and secondary metabolite production. A dramatic advance in Streptomyces research was made recently, by exploiting high-throughput technology to systematically understand RNA levels. In this review, we describe the current status of the system-wide investigation of Streptomyces in terms of RNA, toward expansion of its genetic potential for secondary metabolite synthesis.

Synthetic Biology Approaches in The Development of Engineered Therapeutic Microbes.

Since the intimate relationship between microbes and human health has been uncovered, microbes have been in the spotlight as therapeutic targets for several diseases. Microbes contribute to a wide range of diseases, such as gastrointestinal disorders, diabetes and cancer. However, as host-microbiome interactions have not been fully elucidated, treatments such as probiotic administration and fecal transplantations that are used to modulate the microbial community often cause nonspecific results with serious safety concerns. As an alternative, synthetic biology can be used to rewire microbial networks such that the microbes can function as therapeutic agents. Genetic sensors can be transformed to detect biomarkers associated with disease occurrence and progression. Moreover, microbes can be reprogrammed to produce various therapeutic molecules from the host and bacterial proteins, such as cytokines, enzymes and signaling molecules, in response to a disturbed physiological state of the host. These therapeutic treatment systems are composed of several genetic parts, either identified in bacterial endogenous regulation systems or developed through synthetic design. Such genetic components are connected to form complex genetic logic circuits for sophisticated therapy. In this review, we discussed the synthetic biology strategies that can be used to construct engineered therapeutic microbes for improved microbiome-based treatment.

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins.

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

A study on the non-monophyletic genera Australothrix and Holostichides based on multigene and morphological analyses with a reexamination of type materials (Protozoa: Ciliophora).

In a study of ciliate diversity, we collected two new species of the genera Australothrix and Holostichides. Based on nuclear ribosomal genes, our study shows that these two genera are genetically non-monophyletic. To clarify the issues of the non-monophyly, we reexamined type material of H. heterotypicus, H. terrae, and Birojimia soyaensis. Based on multigene and morphometric analyses, Australothrix and Holostichides are clearly non-monophyletic even in mitochondrial CO1 gene trees. The multigene analyses show a clade composed of A. lineae sp. nov., H. terrae, and B. soyaensis, suggesting that a cytopharynx with argyrophilic structures might be their synapomorphy. A list of species with this type of cytopharynx is provided for further studies to either accept or reject this hypothesis. Based on the reexamination, we discriminate the morphologically nearly identical (cryptic/sibling) species H. obliquocirratus sp. nov. from H. heterotypicus and they show significant genetic dissimilarities in the multigene trees. They showed only few morphological (non-quantitative) differences and thus distinguishing them morphologically needs careful investigation.

Morphology and Molecular Phylogeny of Two New Terrestrial Ciliates, Australocirrus rubrus n. sp. and Notohymena gangwonensis n. sp. (Ciliophora: Oxytrichidae), from South Korea.

Two new soil oxytrichids, Australocirrus rubrus n. sp. and Notohymena gangwonensis n. sp., were discovered from South Korea. Morphologically, A. rubrus shares many features with A. australis, and these two species form a single clade in a molecular tree based on nuclear small subunit ribosomal RNA (SSU rRNA) gene sequences. Australocirrus rubrus mainly differs from A. australis in the color (citrine color vs. reddish) and distribution of the cortical granules. Additionally, we confirm that the genus Australocirrus is not a monophyletic group, as A. shii is separated from the clade comprising the other Australocirrus species, being clustered instead with other taxa. Notohymena gangwonensis n. sp. mainly differs from its congeners by the following combination of features: irregularly distributed cortical granules (vs. arranged in groups associated with cirri and dorsal kineties), variable four or five (usually four) transverse cirri (vs. invariable five), and the anteriormost pretransverse cirrus V/2 on 13.2-16.1% of cell length (vs. on or above 18.9% of cell length). Currently, there are no available gene sequences for members of the genus Notohymena, thus we provide SSU rRNA gene sequences from the new species of Notohymena, as well as detailed morphological descriptions of the novel species.

SoxF Transcription Factors Are Positive Feedback Regulators of VEGF Signaling.

RATIONALE: Vascular endothelial growth factor (VEGF) signaling is a key pathway for angiogenesis and requires highly coordinated regulation. Although the Notch pathway-mediated suppression of excessive VEGF activity via negative feedback is well known, the positive feedback control for augmenting VEGF signaling remains poorly understood. Transcription factor Sox17 is indispensable for angiogenesis, but its association with VEGF signaling is largely unknown. The contribution of other Sox members to angiogenesis also remains to be determined. OBJECTIVE: To reveal the genetic interaction of Sox7, another Sox member, with Sox17 in developmental angiogenesis and their functional relationship with VEGF signaling. METHODS AND RESULTS: Sox7 is expressed specifically in endothelial cells and its global and endothelial-specific deletion resulted in embryonic lethality with severely impaired angiogenesis in mice, substantially overlapping with Sox17 in both expression and function. Interestingly, compound heterozygosity for Sox7 and Sox17 phenocopied vascular defects of Sox7 or Sox17 homozygous knockout, indicating that the genetic cooperation of Sox7 and Sox17 is sensitive to their combined gene dosage. VEGF signaling upregulated both Sox7 and Sox17 expression in angiogenesis via mTOR pathway. Furthermore, Sox7 and Sox17 promoted VEGFR2 (VEGF receptor 2) expression in angiogenic vessels, suggesting a positive feedback loop between VEGF signaling and SoxF. CONCLUSIONS: Our findings demonstrate that SoxF transcription factors are indispensable players in developmental angiogenesis by acting as positive feedback regulators of VEGF signaling.

Morphology and Molecular Phylogeny of Two New Ciliates, Holostichides heterotypicus n. sp. and Holosticha muuiensis n. sp. (Ciliophora: Urostylida).

Two new urostylid species, Holostichides heterotypicus n. sp. and Holosticha muuiensis n. sp., were discovered in South Korea. Morphological and phylogenetic analyses were carried out to confirm that these species are new to science. Holostichides heterotypicus is mainly characterized by the following combination of features: 110-205 µm long in vivo; 5-10 frontoterminal cirri; 6-8 midventral pairs with 2-3 midventral cirral rows; cortical granules present; four bipolar dorsal kineties; and 6-9 caudal cirri. Ontogenetic features of H. heterotypicus are similar to those of H. typicus. Phylogenetic analyses revealed that H. heterotypicus was distantly separated from bakuellid genera Apobakuella, Bakuella, Metaurostylopsis, and Neobakuella. This result is supported by the following features: transverse cirri (present in the other four bakuellids vs. absent in Holostichides) and caudal cirri (absent in the other four bakuellids vs. present in Holostichides). Holosticha muuiensis n. sp. is mainly distinguished from its congeners by the following combination of features: 100-185 long in vivo; shortened undulating membrane; cortical granules lacking; contractile vacuole absent; 51-66 adoral zone of membranelles; 42-60 macronuclear nodules; and five bipolar dorsal kineties. In the phylogenetic tree, Holosticha muuiensis n. sp. clustered with a Holosticha group (containing Holosticha diademata, Holosticha foissneri, and Holosticha heterofoissneri).

A nationwide study of the incidence rate of herb-induced liver injury in Korea.

Discrepant incidence has been reported regarding the incidence of herb-induced liver injury (HILI). To address the growing worldwide concern of HILI, we evaluated the risk of HILI in a nationwide prospective study. Between April 2013 and January 2016, 1001 inpatients (360 males and 641 females) from 10 tertiary hospitals throughout South Korea were treated with herbal drugs and had their liver enzymes periodically measured. A total of six patients met the criteria for HILI with RUCAM scores ranging from 4 to 7. All these participants were women and developed the hepatocellular type of HILI. One HILI participant met the criteria for Hy's law; however, none of six cases presented clinical symptoms related to liver injury. This is the first nationwide prospective study that estimated the extent of the incidence of HILI [total: 0.60%, 95% confidence interval (CI) 0.12-1.08; women: 0.95%, 95% CI 0.19-1.68] and described its features in hospitalized participants.

Notch pathway targets proangiogenic regulator Sox17 to restrict angiogenesis.

RATIONALE: The Notch pathway stabilizes sprouting angiogenesis by favoring stalk cells over tip cells at the vascular front. Because tip and stalk cells have different properties in morphology and function, their transcriptional regulation remains to be distinguished. Transcription factor Sox17 is specifically expressed in endothelial cells, but its expression and role at the vascular front remain largely unknown. OBJECTIVE: To specify the role of Sox17 and its relationship with the Notch pathway in sprouting angiogenesis. METHODS AND RESULTS: Endothelial-specific Sox17 deletion reduces sprouting angiogenesis in mouse embryonic and postnatal vascular development, whereas Sox17 overexpression increases it. Sox17 promotes endothelial migration by destabilizing endothelial junctions and rearranging cytoskeletal structure and upregulates expression of several genes preferentially expressed in tip cells. Interestingly, Sox17 expression is suppressed in stalk cells in which Notch signaling is relatively high. Notch activation by overexpressing Notch intracellular domain reduces Sox17 expression both in primary endothelial cells and in retinal angiogenesis, whereas Notch inhibition by delta-like ligand 4 (Dll4) blockade increases it. The Notch pathway regulates Sox17 expression mainly at the post-transcriptional level. Furthermore, endothelial Sox17 ablation rescues vascular network from excessive tip cell formation and hyperbranching under Notch inhibition in developmental and tumor angiogenesis. CONCLUSIONS: Our findings demonstrate that the Notch pathway restricts sprouting angiogenesis by reducing the expression of proangiogenic regulator Sox17.

CRISPRi-Driven Genetic Screening for Designing Novel Microbial Phenotypes.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has enabled rapid advances in genomic engineering and transcriptional regulation. Specifically, CRISPR interference (CRISPRi) system has been used to systematically investigate the gene functions of microbial strains in a high-throughput manner. This method involves growth profiling using cells that have been transformed with the deactivated Cas9 (dCas9) and single-guide RNA (sgRNA) libraries that target individual genes. The fitness scores of each gene are calculated by measuring the abundance of individual sgRNAs during cell growth and represent gene essentiality. In this chapter, a process is described for functional genetic screening using CRISPRi at the whole-genome scale, starting from the synthesis of sgRNA libraries, construction of CRISPRi libraries, and identification of essential genes through growth profiling. The commensal bacterium Bacteroides thetaiotaomicron was used to implement the protocol. This method is expected to be applicable to a broader range of microorganisms to explore the novel phenotypic characteristics of microorganisms.

Reduction-to-synthesis: the dominant approach to genome-scale synthetic biology.

Advances in systems and synthetic biology have propelled the construction of reduced bacterial genomes. Genome reduction was initially focused on exploring properties of minimal genomes, but more recently it has been deployed as an engineering strategy to enhance strain performance. This review provides the latest updates on reduced genomes, focusing on dual-track approaches of top-down reduction and bottom-up synthesis for their construction. Using cases from studies that are based on established industrial workhorse strains, we discuss the construction of a series of synthetic phenotypes that are candidates for biotechnological applications. Finally, we address the possible uses of reduced genomes for biotechnological applications and the needed future research directions that may ultimately lead to the total synthesis of rationally designed genomes.

Automatic Quantitative Assessment for Diagnostic and Therapeutic Response in Rodent Myocardial Infarct Model.

The purpose of this study was to investigate the most appropriate methodological approach for the automatic measurement of rodent myocardial infarct polar map using histogram-based thresholding and unsupervised deep learning (DL)-based segmentation. A rat myocardial infarction model was induced by ligation of the left coronary artery. Positron emission tomography (PET) was performed 60 min after the administration of 18F-fluoro-deoxy-glucose (18F-FDG), and PET was performed after injecting 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone). Single photon emission computed tomography was performed 60 min after injection of 99mTc-hexakis-2-methoxyisobutylisonitrile and 201Tl. Delayed contrast-enhanced magnetic resonance imaging was performed after injecting Gd-DTPA-BMA. Three types of thresholding methods (naive thresholding, Otsu's algorithm, and multi-Gaussian mixture model (MGMM)) were used. DL segmentation methods were based on a convolution neural network and trained with constraints on feature similarity and spatial continuity of the response map extracted from images by the network. The relative infarct sizes measured by histology and estimated R2 for 18F-FDG were 0.8477, 0.7084, 0.8353, and 0.9024 for naïve thresholding, Otsu's algorithm, MGMM, and DL segmentation, respectively. DL-based method improved the accuracy of MI size assessment.

Defect-Engineered Semiconducting van der Waals Thin Film at Metal-Semiconductor Interface of Field-Effect Transistors.

The significance of metal-semiconductor interfaces and their impact on electronic device performance have gained increasing attention, with a particular focus on investigating the contact metal. However, another avenue of exploration involves substituting the contact metal at the metal-semiconductor interface of field-effect transistors with semiconducting layers to introduce additional functionalities to the devices. Here, a scalable approach for fabricating metal-oxide-semiconductor (channel)-semiconductor (interfacial layer) field-effect transistors is proposed by utilizing solution-processed semiconductors, specifically semiconducting single-walled carbon nanotubes and molybdenum disulfide, as the channel and interfacial semiconducting layers, respectively. The work function of the interfacial MoS2 is modulated by controlling the sulfur vacancy concentration through chemical treatment, which results in distinctive energy band alignments within a single device configuration. The resulting band alignments lead to multiple functionalities, including multivalued transistor characteristics and multibit nonvolatile memory (NVM) behavior. Moreover, leveraging the stable NVM properties, we demonstrate artificial synaptic devices with 88.9% accuracy of MNIST image recognition.

AXL/WRNIP1 Mediates Replication Stress Response and Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer.

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.