Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli.

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Identification of Mitral Valve Prolapse on Non-electrocardiography-gated Enhanced Chest Computed Tomography.

PURPOSE: The primary imaging modality for the diagnosis of mitral valve prolapse (MVP) is echocardiography supplemented by electrocardiography (ECG)-gated cardiac computed tomography (CT) angiography. However, we have recently encountered patients with MVP who were initially identified on non-ECG-gated enhanced chest CT. The purpose of this study is to evaluate the diagnostic accuracy of non-ECG-gated enhanced chest CT to predict the presence of MVP. PATIENTS AND METHODS: Of 92 patients (surgically confirmed MVP who underwent non-ECG-gated chest CT), 27 patients were excluded for motion artifact or insufficient surgical correlation, and 65 patients were ultimately included. As a control, 65 patients with dyspnea and without MVP (non-ECG-gated chest CT and echocardiography were performed within 1 month) were randomly selected. We retrospectively analyzed an asymmetric double line sign on axial CT images for the presence of MVP. The asymmetric double line sign was defined as the presence of a linear structure, not located in the plane traversing the mitral annulus. RESULTS: Use of the asymmetric double line sign to predict MVP on non-ECG-gated CT showed modest sensitivity, high specificity, modest negative predictive value, and high positive predictive value of 59% (38/65), 99% (64/65), 70% (64/91), and 97% (38/39), respectively. CONCLUSION: The asymmetric double line sign on non-ECG-gated enhanced chest CT may be a valuable finding to predict the presence of MVP. Familiarity with this CT finding may lead to prompt diagnosis and proper management of MVP.

Effects of klotho protein or klotho knockdown in porcine oocytes at different stages.

Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo. However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.

Efficient reverse genetics approach involving infectious subgenomic amplicon for engineering dengue virus.

Dengue virus, which belongs to the Flaviviridae family, can induce a range of symptoms from mild to severe, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. While infectious cloning technology is a useful tool for understanding viral pathogenesis and symptoms, it exhibits limitations when constructing the entire Flavivirus genome. The instability and toxicity of the genome to bacteria make its full-length construction in bacterial vectors a time-consuming and laborious process. To address these challenges, we employed the modified infectious subgenomic amplicon (ISA) method in this study, which can potentially be a superior tool for reverse genetic studies on the dengue virus. Using ISA, we generated recombinant dengue viruses de novo and validated their robust replication in both human and insect cell lines, which was comparable to that of the original strains. Moreover, the efficiency of ISA in genetically modifying the dengue virus was elucidated by successfully inserting the gene for green fluorescence protein into the genome of dengue virus serotype 4. Overall, this study highlighted the effectiveness of ISA for genetically engineering the dengue virus and provided a technical basis for a convenient reverse genetics system that could expedite investigations into the dengue virus.

HMGB1, a potential regulator of tumor microenvironment in KSHV-infected endothelial cells.

High-mobility group box 1 (HMGB1) is a protein that binds to DNA and participates in various cellular processes, including DNA repair, transcription, and inflammation. It is also associated with cancer progression and therapeutic resistance. Despite its known role in promoting tumor growth and immune evasion in the tumor microenvironment, the contribution of HMGB1 to the development of Kaposi's sarcoma (KS) is not well understood. We investigated the effect of HMGB1 on KS pathogenesis using immortalized human endothelial cells infected with Kaposi's sarcoma-associated human herpes virus (KSHV). Our results showed that a higher amount of HMGB1 was detected in the supernatant of KSHV-infected cells compared to that of mock-infected cells, indicating that KSHV infection induced the secretion of HMGB1 in human endothelial cells. By generating HMGB1 knockout clones from immortalized human endothelial cells using CRISPR/Cas9, we elucidated the role of HMGB1 in KSHV-infected endothelial cells. Our findings indicate that the absence of HMGB1 did not induce lytic replication in KSHV-infected cells, but the cell viability of KSHV-infected cells was decreased in both 2D and 3D cultures. Through the antibody array for cytokines and growth factors, CXCL5, PDGF-AA, G-CSF, Emmprin, IL-17A, and VEGF were found to be suppressed in HMGB1 KO KSHV-infected cells compared to the KSHV-infected wild-type control. Mechanistically, phosphorylation of p38 would be associated with transcriptional regulation of CXCL5, PDGF-A and VEGF. These observations suggest that HMGB1 may play a critical role in KS pathogenesis by regulating cytokine and growth factor secretion and emphasize its potential as a therapeutic target for KS by modulating the tumor microenvironment.

Medical education program implementation experience in an elective course: a case study in South Korea.

PURPOSE: This study was conducted to examine the results of designing and implementing a teaching program for medical education as the elective course for 4th-year students of medical course. METHODS: In order to design the teaching program for medical education as an elective course, we conducted literature review, five medical education experts were interviewed, and the literature required in the design process was reviewed. A developing teaching program was implemented as an elective course in a medical school of Korea, and 4th-year students of medical course participated in the program. RESULTS: In the elective course, the medical education program process competencies were derived into three categories: theoretical educational knowledge, teaching competency, and research competency for education. Moreover, instructional materials were developed to help students achieve these competencies. And project-based learning strategy was selected and implemented for 4th-year students in medical course, and positive satisfaction was confirmed. CONCLUSION: As a study designed and implemented in a medical education program in a medical school in Korea, it is expected to be helpful when introducing medical education to undergraduate students or developing a medical education program to strengthen the teaching capacity of residents.

Endolysosomal impairment by binding of amyloid beta or MAPT/Tau to V-ATPase and rescue via the HYAL-CD44 axis in Alzheimer disease.

Impaired activities and abnormally enlarged structures of endolysosomes are frequently observed in Alzheimer disease (AD) brains. However, little is known about whether and how endolysosomal dysregulation is triggered and associated with AD. Here, we show that vacuolar ATPase (V-ATPase) is a hub that mediates proteopathy of oligomeric amyloid beta (Aß) and hyperphosphorylated MAPT/Tau (p-MAPT/Tau). Endolysosomal integrity was largely destroyed in Aß-overloaded or p-MAPT/Tau-positive neurons in culture and AD brains, which was a necessary step for triggering neurotoxicity, and treatments with acidic nanoparticles or endocytosis inhibitors rescued the endolysosomal impairment and neurotoxicity. Interestingly, we found that the lumenal ATP6V0C and cytosolic ATP6V1B2 subunits of the V-ATPase complex bound to the internalized Aß and cytosolic PHF-1-reactive MAPT/Tau, respectively. Their interactions disrupted V-ATPase activity and accompanying endolysosomal activity in vitro and induced neurodegeneration. Using a genome-wide functional screen, we isolated a suppressor, HYAL (hyaluronidase), which reversed the endolysosomal dysfunction and proteopathy and alleviated the memory impairment in 3xTg-AD mice. Further, we found that its metabolite hyaluronic acid (HA) and HA receptor CD44 attenuated neurotoxicity in affected neurons via V-ATPase. We propose that endolysosomal V-ATPase is a bona fide proteotoxic receptor that binds to pathogenic proteins and deteriorates endolysosomal function in AD, leading to neurodegeneration in proteopathy.Abbreviations: AAV, adeno-associated virus; Aß, amyloid beta; AD, Alzheimer disease; APP, amyloid beta precursor protein; ATP6V0C, ATPase H+ transporting V0 subunit c; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1B2, ATPase H+ transporting V1 subunit B2; CD44.Fc, CD44-mouse immunoglobulin Fc fusion construct; Co-IP, co-immunoprecipitation; CTSD, cathepsin D; HA, hyaluronic acid; HMWHA, high-molecular-weight hyaluronic acid; HYAL, hyaluronidase; i.c.v, intracerebroventricular; LMWHA, low-molecular-weight hyaluronic acid; NPs, nanoparticles; p-MAPT/Tau, hyperphosphorylated microtubule associated protein tau; PI3K, phosphoinositide 3-kinase; V-ATPase, vacuolar-type H+-translocating ATPase; WT, wild-type.

CXCL1 confers a survival advantage in Kaposi's sarcoma-associated herpesvirus-infected human endothelial cells through STAT3 phosphorylation.

Many cytokines produced by Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells have been shown to participate in the pathogenesis of KSHV. Determination of the exact role of cytokines in Kaposi's sarcoma (KS) pathogenesis is limited, however, by the difficulty to manipulate the target genes in human endothelial cells. In this study, we sought to elucidate the role of cytokines in KSHV-infected human immortalized endothelial cell line (HuARLT cells) by knockout (KO) of the corresponding target genes using the CRISPR/Cas9 system. The cytokine production profile of KSHV-infected HuARLT cells was analyzed using a protein array, and several cytokines were found to be highly upregulated following KSHV infection. This study focused on CXCL1, which was investigated by knocked out in HuARLT cells. KSHV-infected CXCL1 KO cells underwent increased cell death compared to KSHV-infected wild-type (WT) cells and mock-infected CXCL1 KO cells. Lytic replication was not observed in KSHV-infected WT nor CXCL1 KO cells. Phosphorylation of STAT3 was significantly suppressed in KSHV-infected CXCL1 KO cells. Additionally, inhibitors of STAT3 and CXCL1 induced cell death in KSHV-infected endothelial cells. Our results show that CXCL1 production is required for the survival of KSHV-infected endothelial cells, and the CXCL1 to STAT3 phosphorylation signaling pathway may be a therapeutic target for KS.

High-throughput genetic engineering tools for regulating gene expression in a microbial cell factory.

With the rapid advances in biotechnological tools and strategies, microbial cell factory-constructing strategies have been established for the production of value-added compounds. However, optimizing the tradeoff between the biomass, yield, and titer remains a challenge in microbial production. Gene regulation is necessary to optimize and control metabolic fluxes in microorganisms for high-production performance. Various high-throughput genetic engineering tools have been developed for achieving rational gene regulation and genetic perturbation, diversifying the cellular phenotype and enhancing bioproduction performance. In this paper, we review the current high-throughput genetic engineering tools for gene regulation. In particular, technological approaches used in a diverse range of genetic tools for constructing microbial cell factories are introduced, and representative applications of these tools are presented. Finally, the prospects for high-throughput genetic engineering tools for gene regulation are discussed.

Identification of a gene cluster for D-tagatose utilization in Escherichia coli B2 phylogroup.

D-Tagatose is a promising low-calorie sugar-substituting sweetener in the food industry. Most ingested D-tagatose is fermented by intestinal microorganisms. Until now, Escherichia coli has been considered incapable of growing on D-tagatose. Here, we discovered a gene cluster involved in D-tagatose utilization in E. coli. The chromosome of the intestinal probiotic E. coli Nissle 1917 contains a six-gene cluster encoding the ABC transporter, D-tagatose kinase, D-tagatose-bisphosphate aldolase, and putative aldose 1-epimerase. The functionality of the gene cluster was experimentally validated. Based on single-gene deletions, D-tagatose dissimilation occurs via D-tagatose 6-phosphate to D-tagatose 1,6-bisphosphate to D-glyceraldehyde 3-phosphate plus dihydroxyacetone phosphate. Remarkably, this gene cluster was located in 93% of the completely sequenced genomes of the E. coli B2 phylogroup, which contains the majority of extraintestinal pathogenic and adherent-invasive E. coli strains prevalent in patients with inflammatory bowel disease. This highlights the importance of understanding the clinical significance of D-tagatose in microbiota alterations.

Electrochemiluminescence Systems for the Detection of Biomarkers: Strategical and Technological Advances.

Electrochemiluminescence (ECL)-based sensing systems rely on light emissions from luminophores, which are generated by high-energy electron transfer reactions between electrogenerated species on an electrode. ECL systems have been widely used in the detection and monitoring of diverse, disease-related biomarkers due to their high selectivity and fast response times, as well as their spatial and temporal control of luminance, high controllability, and a wide detection range. This review focuses on the recent strategic and technological advances in ECL-based biomarker detection systems. We introduce several sensing systems for medical applications that are classified according to the reactions that drive ECL signal emissions. We also provide recent examples of sensing strategies and technologies based on factors that enhance sensitivity and multiplexing abilities as well as simplify sensing procedures. This review also discusses the potential strategies and technologies for the development of ECL systems with an enhanced detection ability.

Colorimetric Systems for the Detection of Bacterial Contamination: Strategy and Applications.

Bacterial contamination is a public health concern worldwide causing enormous social and economic losses. For early diagnosis and adequate management to prevent or treat pathogen-related illnesses, extensive effort has been put into the development of pathogenic bacterial detection systems. Colorimetric sensing systems have attracted increasing attention due to their simple and single-site operation, rapid signal readout with the naked eye, ability to operate without external instruments, portability, compact design, and low cost. In this article, recent trends and advances in colorimetric systems for the detection and monitoring of bacterial contamination are reviewed. This article focuses on pathogen detection strategies and technologies based on reaction factors that affect the color change for visual readout. Reactions used in each strategy are introduced by dividing them into the following five categories: external pH change-induced pH indicator reactions, intracellular enzyme-catalyzed chromogenic reactions, enzyme-like nanoparticle (NP)-catalyzed substrate reactions, NP aggregation-based reactions, and NP accumulation-based reactions. Some recently developed colorimetric systems are introduced, and their challenges and strategies to improve the sensing performance are discussed.

Synthetic fused sRNA for the simultaneous repression of multiple genes.

Efficient control over multiple gene expression still presents a major challenge. Synthetic sRNA enables targeted gene expression control in trans without directly modifying the chromosome, but its use to simultaneously target multiple genes can often cause cell growth defects because of the need for additional energy for transcription and lowering of their repression efficiency by limiting the amount of Hfq protein. To address these limitations, we present fusion sRNA (fsRNA) that simultaneously regulates the translation of multiple genes efficiently. It is constructed by linking the mRNA-binding modules for multiple targeted genes in one sRNA scaffold via one-pot generation using overlap extension PCR. The repression capacity of fsRNA was demonstrated by the construction of sRNAs to target four endogenous genes: caiF, hybG, ytfR and minD in Escherichia coli. Their cross-reactivity and the effect on cell growth were also investigated. As practical applications, we applied fsRNA to violacein- and protocatechuic acid-producing strains, resulting in increases of 13% violacein and 81% protocatechuic acid, respectively. The developed fsRNA-mediated multiple gene expression regulation system thus enables rapid and efficient development of optimised cell factories for valuable chemicals without cell growth defects and limiting cellular resources.Key points• Synthetic fusion sRNA (fsRNA)-based system was constructed for the repression of multiple target genes.• fsRNA repressed multiple genes by only expressing a single sRNA while minimising the cellular burden.• The application of fsRNA showed the increased production titers of violacein (13%) and protocatechuic acid (81%).

Construction of a tunable promoter library to optimize gene expression in Methylomonas sp. DH-1, a methanotroph, and its application to cadaverine production.

BACKGROUND: As methane is 84 times more potent than carbon dioxide in exacerbating the greenhouse effect, there is an increasing interest in the utilization of methanotrophic bacteria that can convert harmful methane into various value-added compounds. A recently isolated methanotroph, Methylomonas sp. DH-1, is a promising biofactory platform because of its relatively fast growth. However, the lack of genetic engineering tools hampers its wide use in the bioindustry. RESULTS: Through three different approaches, we constructed a tunable promoter library comprising 33 promoters that can be used for the metabolic engineering of Methylomonas sp. DH-1. The library had an expression level of 0.24-410% when compared with the strength of the lac promoter. For practical application of the promoter library, we fine-tuned the expressions of cadA and cadB genes, required for cadaverine synthesis and export, respectively. The strain with PrpmB-cadA and PDnaA-cadB produced the highest cadaverine titre (18.12 ± 1.06 mg/L) in Methylomonas sp. DH-1, which was up to 2.8-fold higher than that obtained from a non-optimized strain. In addition, cell growth and lysine (a precursor of cadaverine) production assays suggested that gene expression optimization through transcription tuning can afford a balance between the growth and precursor supply. CONCLUSIONS: The tunable promoter library provides standard and tunable components for gene expression, thereby facilitating the use of methanotrophs, specifically Methylomonas sp. DH-1, as a sustainable cell factory.

The Patent Ductus Arteriosus in Adults with Special Focus on Role of CT.

The primary imaging modality for the diagnosis of patent ductus arteriosus (PDA) is echocardiography. However, CT may be the technique on which an incidental PDA is first recognized because of the increasing number of chest CT scans performed for a variety of causes. Identification of PDA on CT may lead to earlier closure using a PDA occluder device. Immediate identification of incidental PDA is important, but a high rate of missed diagnosis of PDA has been reported due to its small size and anatomic location. In addition, echocardiography may overlook the presence of even a large PDA due to decrease in the amount of shunting through the PDA caused by high pulmonary artery pressures. This review provides the basic CT anatomy and clinical perspective of PDA, and discusses the role of CT in the evaluation of PDA as well as methods to avoid overlooking a small PDA on CT.

Diagnostic Accuracy of Coronary Artery Occlusion and Myocardial Perfusion Defect on Non-Gated Enhanced Chest CT in Predicting Acute Myocardial Infarction.

The purpose of this study was to evaluate the diagnostic accuracy of coronary artery occlusion (CAO) and myocardial perfusion defect (MPD) identified on non-gated enhanced chest CT in patients with acute myocardial infarction (AMI). We retrospectively assessed 99 patients with AMI (group 1, n = 33) and without AMI (group 2, n = 66) who underwent non-gated chest CT. We analyzed the presence of MPD and CAO on non-gated chest CT. MPD on the CT was categorized using a three-point scale (0 = no definite MPD; 1 = probable artifact or questionable MPD; 2 = probable MPD). Presence of CAO was defined as an abrupt change of contrast enhancement in a coronary artery segment with no or minimal coronary motion on the CT. There were 42.4% and 12.1% patients with probable MPD (p = 0.002), and 18.2% and 0% patients with CAO (p = 0.001) in groups 1 and 2, respectively. Probable MPD alone and simultaneous presence of CAO and probable MPD to predict AMI resulted in sensitivity, specificity, negative predictive value, and positive predictive valve of 42.4%, 87.9%, 75.3%, and 63.6%, respectively, and 12.1%, 100%, 69.5%, and 100%, respectively. In conclusion, probable MPD alone on non-gated chest CT demonstrated a relatively low sensitivity, high specificity, and modest positive predictive value for the prediction of AMI on non-gated enhanced chest CT. Although it is rare, simultaneous presence of CAO and probable MPD had a high positive predictive value to predict AMI on non-gated enhanced chest CT.

Recent Advances in Micro/Nanomaterial-Based Aptamer Selection Strategies.

Aptamers are artificial nucleic acid ligands that have been employed in various fundamental studies and applications, such as biological analyses, disease diagnostics, targeted therapeutics, and environmental pollutant detection. This review focuses on the recent advances in aptamer discovery strategies that have been used to detect various chemicals and biomolecules. Recent examples of the strategies discussed here are based on the classification of these micro/nanomaterial-mediated systematic evolution of ligands by exponential enrichment (SELEX) platforms into three categories: bead-mediated, carbon-based nanomaterial-mediated, and other nanoparticle-mediated strategies. In addition to describing the advantages and limitations of the aforementioned strategies, this review discusses potential strategies to develop high-performance aptamers.

HMGB1 Knockout Decreases Kaposi's Sarcoma-Associated Herpesvirus Virion Production in iSLK BAC16 Cells by Attenuating Viral Gene Expression.

Multiple host proteins affect the gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) during latent and lytic replication. High-mobility group box 1 (HMGB1) serves as a highly conserved chromosomal protein inside the cell and a prototypical damage-associated molecular pattern molecule outside the cell. HMGB1 has been shown to play a pathogenic role in viral infectious diseases and to regulate the lytic replication of KSHV. However, its functional effects on the KSHV life cycle in KSHV-infected cells have not been fully elucidated. Here, we explored the role of intracellular and extracellular HMGB1 in KSHV virion production by employing CRISPR/Cas9-mediated HMGB1 knockout in the KSHV-producing iSLK BAC16 cell line. Intracellular HMGB1 formed complexes with various proteins, and the abundance of HMGB1-interacting proteins changed during latent and lytic replication. Moreover, extracellular HMGB1 was found to enhance lytic replication by phosphorylating JNK. Of note, the expression of viral genes was attenuated during lytic replication in HMGB1 knockout iSLK BAC16 cells, with significantly decreased production of infectious virions compared to that of wild-type cells. Collectively, our results demonstrate that HMGB1 is an important cellular cofactor that affects the generation of infectious KSHV progeny during lytic replication. IMPORTANCE The high-mobility group box 1 (HMGB1) protein has many intra- and extracellular biological functions with an intricate role in various diseases. In certain viral infections, HMGB1 affects the viral life cycle and pathogenesis. In this study, we explored the effects of HMGB1 knockout on the production of Kaposi's sarcoma-associated herpesvirus (KSHV). HMGB1 knockout decreased virion production in KSHV-producing cells by decreasing the expression of viral genes. The processes by which HMGB1 affects KSHV production may occur inside or outside infected cells. For instance, several cellular and viral proteins interacted with intracellular HMGB1 in a nucleosomal complex, whereas extracellular HMGB1 induced JNK phosphorylation, thereby enhancing lytic replication. Our results suggest that both intracellular and extracellular HMGB1 are necessary for efficient KSHV replication. Thus, HMGB1 may represent an effective therapeutic target for the regulation of KSHV production.

Biosensing Applications Using Nanostructure-Based Localized Surface Plasmon Resonance Sensors.

Localized surface plasmon resonance (LSPR)-based biosensors have recently garnered increasing attention due to their potential to allow label-free, portable, low-cost, and real-time monitoring of diverse analytes. Recent developments in this technology have focused on biochemical markers in clinical and environmental settings coupled with advances in nanostructure technology. Therefore, this review focuses on the recent advances in LSPR-based biosensor technology for the detection of diverse chemicals and biomolecules. Moreover, we also provide recent examples of sensing strategies based on diverse nanostructure platforms, in addition to their advantages and limitations. Finally, this review discusses potential strategies for the development of biosensors with enhanced sensing performance.