A humanized ACE2 mouse model recapitulating age- and sex-dependent immunopathogenesis of COVID-19.

In the ongoing battle against coronavirus disease 2019 (COVID-19), understanding its pathogenesis and developing effective treatments remain critical challenges. The creation of animal models that closely replicate human infection stands as a critical step forward in this research. Here, we present a genetically engineered mouse model with specifically-humanized knock-in ACE2 (hiACE2) receptors. This model, featuring nine specific amino acid substitutions for enhanced interaction with the viral spike protein, enables efficient severe acute respiratory syndrome coronavirus 2 replication in respiratory organs without detectable infection in the central nervous system. Moreover, it mirrors the age- and sex-specific patterns of morbidity and mortality, as well as the immunopathological features observed in human COVID-19 cases. Our findings further demonstrate that the depletion of eosinophils significantly reduces morbidity and mortality, depending on the infecting viral dose and the sex of the host. This reduction is potentially achieved by decreasing the pathogenic contribution of eosinophil-mediated inflammation, which is strongly correlated with neutrophil activity in human patients. This underscores the model's utility in studying the immunopathological aspects of COVID-19 and represents a significant advancement in COVID-19 modeling. It offers a valuable tool for testing vaccines and therapeutics, enhancing our understanding of the disease mechanisms and potentially guiding more targeted and effective treatments.

Exploring 2-mercapto-N-arylacetamide analogs as promising anti-melanogenic agents: in vitro and in vivo evaluation.

Based on the hypothesis that the 2-mercaptoacetamide moiety chelates the copper ions of tyrosinase, 2-mercapto-N-arylacetamide (2-MAA) analogs were designed and synthesized as potential tyrosinase inhibitors. Four 2-MAA analogs showed low IC50 values ranging from 0.95 to 2.0 µM against mushroom tyrosinase, which was 12-26 times lower than that of kojic acid (IC50 value = 24.3 µM). However, according to a copper ion chelation experiment performed, the 2-MAA analogs did not participate in chelation with copper ions. To identify the mode of inhibition of the 2-MAA analogs, kinetic studies were performed, and the results were supported by docking results. In addition, docking simulation results suggested that the 2-MAA analogs strongly inhibited tyrosinase activity because of the hydrogen bonding of the amide NH group and the hydrophobic interaction of the aryl ring instead of chelation with copper ions. In experiments using B16F10 cells, 2-MAA analogs were shown to inhibit melanin production by inhibiting cellular tyrosinase activity. Western blotting showed that in addition to directly inhibiting tyrosinase activity, analog 7 also has an anti-melanogenic effect by inhibiting the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. The 2-MAA analogs showed no appreciable cytotoxicity against HaCaT and B16F10 cells, making them suitable for dermal applications. In a depigmentation experiment using zebrafish embryos, analogs 1 and 2 showed more potent depigmentation effects than kojic acid even at 1000 times lower concentration than that of kojic acid. These results suggest that the 2-MAA analogs are promising anti-melanogenic agents that can inhibit most tyrosinases in various species.

Plasma Kallistatin and Progranulin as Predictive Biomarkers of Intraamniotic Inflammation, Microbial Invasion of the Amniotic Cavity, and Composite Neonatal Morbidity/Mortality in Women With Preterm Premature Rupture of Membranes.

PROBLEM: To explore the clinical utility of nine inflammatory immune-, adhesion-, and extracellular matrix-related mediators in the plasma for predicting intraamniotic inflammation and/or microbial invasion of the amniotic cavity (IAI/MIAC) and composite neonatal morbidity and/or mortality (CNMM) in women with preterm premature rupture of membranes (PPROM) when used alone or in combination with conventional blood-, ultrasound-, and clinical-based factors. METHODS OF STUDY: This retrospective cohort comprised 173 singleton pregnant women with PPROM (24 + 0 - 33 + 6 weeks), who underwent amniocentesis. Amniotic fluid was cultured for microorganisms and assayed for IL-6 levels. Plasma levels of AFP, CXCL14, E-selectin, Gal-3BP, kallistatin, progranulin, P-selectin, TGFBI, and VDBP were determined by ELISA. Ultrasonographic cervical length (CL) and neutrophil-to-lymphocyte ratio (NLR) were measured. RESULTS: Multivariate logistic regression analyses revealed significant associations between (i) decreased plasma kallistatin levels and IAI/MIAC and (ii) decreased plasma progranulin levels and increased CNMM risk after adjusting for baseline variables (e.g., gestational age at sampling [or delivery] and parity). Using stepwise regression analysis, noninvasive prediction models for IAI/MIAC and CNMM risks were developed, which included plasma progranulin levels, NLR, CL, and gestational age at sampling, and provided a good prediction of the corresponding endpoints (area under the curve: 0.79 and 0.87, respectively). CONCLUSIONS: Kallistatin and progranulin are potentially valuable plasma biomarkers for predicting IAI/MIAC and CNMM in women with PPROM. Particularly, the combination of these plasma biomarkers with conventional blood-, ultrasound-, and clinical-based factors can significantly support the diagnosis of IAI/MIAC and CNMM.

Mining biosynthetic gene clusters in Paenibacillus genomes to discover novel antibiotics.

BACKGROUND: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied. RESULTS: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin. CONCLUSIONS: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.

Identification and characterization of plasma proteins associated with intra-amniotic inflammation and/or infection in women with preterm labor.

This study aimed to identify plasma proteins that could serve as potential biomarkers for microbial invasion of the amniotic cavity (MIAC) or intra-amniotic inflammation (IAI) in women with preterm labor (PTL). A retrospective cohort comprised singleton pregnant women with PTL (24-34 weeks) who underwent amniocentesis. Pooled plasma samples were analyzed by label-free liquid chromatography-tandem mass spectrometry for proteome profiling in a nested case-control study (concomitant MIAC/IAI cases vs. non-MIAC/IAI controls [n = 10 per group]). Eight target proteins associated with MIAC/IAI were further verified by immunoassays in a large cohort (n = 230). Shotgun proteomic analysis revealed 133 differentially expressed proteins (fold change > 1.5, P < 0.05) in the plasma of MIAC/IAI cases. Further quantification confirmed that the levels of AFP were higher and those of kallistatin and TGFBI were lower in the plasma of women with MIAC and that the levels of kallistatin and TGFBI were lower in the plasma of women with IAI than in those without these conditions. The area under the curves of plasma AFP, kallistatin, and TGFBI ranged within 0.67-0.81 with respect to each endpoint. In summary, plasma AFP, kallistatin, and TGFBI may represent valuable non-invasive biomarkers for predicting MIAC or IAI in women with PTL.

Vaccine Development for Severe Fever with Thrombocytopenia Syndrome Virus in Dogs.

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening viral zoonosis. The causative agent of this disease is the Dabie bandavirus, which is usually known as the SFTS virus (SFTSV). Although the role of vertebrates in SFTSV transmission to humans remains uncertain, some reports have suggested that dogs could potentially transmit SFTSV to humans. Consequently, preventive measures against SFTSV in dogs are urgently needed. In the present study, dogs were immunized three times at two-week intervals with formaldehyde-inactivated SFTSV with two types of adjuvants. SFTSV (KCD46) was injected into all dogs two weeks after the final immunization. Control dogs showed viremia from 2 to 4 days post infection (dpi), and displayed white pulp atrophy in the spleen, along with a high level of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay (TUNEL) positive area. However, the inactivated SFTSV vaccine groups exhibited rare pathological changes and significantly reduced TUNEL positive areas in the spleen. Furthermore, SFTSV viral loads were not detected at any of the tested dpi. Our results indicate that both adjuvants can be safely used in combination with an inactivated SFTSV formulation to induce strong neutralizing antibodies. Inactivated SFTSV vaccines effectively prevent pathogenicity and viremia in dogs infected with SFTSV. In conclusion, our study highlighted the potential of inactivated SFTSV vaccination for SFTSV control in dogs.

Comparative analysis of the efficacy of vaccines using structural protein subunits of the severe fever with thrombocytopenia syndrome virus.

The severe fever with thrombocytopenia syndrome virus (SFTSV) represents a significant emerging health threat as a tick-borne pathogen that causes SFTS, with mortality rates ranging between 10 and 30%. Despite the considerable risk presented by SFTSV, an effective vaccine has yet to be developed. Our study assessed the efficacy of recombinant protein vaccines, focusing on the purified nucleocapsid protein (NP) and surface glycoproteins (Gn and Gc), against SFTSV in both singular and combined formulations. Individual vaccinations with NP or Gn subunits yielded partial protection in type I interferon receptor-knockout (IFNAR-KO) mice, with survival rates of 66.7 and 16.7%, respectively, whereas Gc vaccination did not confer significant protection, resulting in 100% mortality similar to that of the unvaccinated control group. Notably, NP vaccination substantially enhanced antigen-specific T cell responses, and Gc vaccination exhibited strong neutralizing activity against SFTSV. Among the combined recombinant protein formulations (Gn + NP, Gc + NP, and Gn + Gc + NP) tested, the Gc + NP combination provided the highest survival rate (85.7%) following challenge with a lethal dose of SFTSV, highlighting its potential as a vaccine candidate. Longitudinal studies showed that antibody levels in both wild type C57BL/6 and IFNAR-KO mice peaked between 2 and 3 months post-vaccination and declined over time. A notable decrease in NP-specific CD8+ T cell responses was observed 6 months post-vaccination in C57BL/6 mice, while NP-specific CD4+ T cell responses persisted up to 12 months. By 12 months post-vaccination, all IFNAR-KO mice vaccinated with single subunit antigens succumbed to the virus, suggesting that effective protection against SFTS may rely on antibody responses to subunit antigens and/or CD8+ T cell activity. These findings underscore the necessity of an optimized SFTS vaccine that combines protective antigens with an adjuvant system to ensure durable humoral and cellular immunity.

Assessment of acetylcholinesterase activity in CD9-positive exosomes from patients with Parkinson's disease.

Introduction: Parkinson's disease (PD) is a neurodegenerative disorder characterized by dopaminergic dysfunction and associated with abnormalities in the cholinergic system. However, the relationship between PD and cholinergic dysfunction, particularly in exosomes, is not fully understood. Methods: We enrolled 37 patients with PD and 44 healthy controls (HC) to investigate acetylcholinesterase (AChE) activity in CD9-positive and L1CAM-positive exosomes. Exosomes were isolated from plasma using antibody-coupled magnetic beads, and their sizes and concentrations were assessed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Subsequently, the AChE activity in these exosomes was analyzed in relation to various clinical parameters. Results: A significant decrease in AChE activity was observed in CD9-positive exosomes derived from patients with PD, whereas no significant differences were found in L1CAM-positive exosomes. Further analysis with a larger sample size confirmed a substantial reduction in AChE activity in CD9-positive exosomes from the PD plasma, with moderate diagnostic accuracy. The decrease in AChE activity of CD9-positive exosomes did not show an association with cognitive impairment but displayed a trend toward correlation with PD progression. Discussion: The reduction in AChE activity in CD9-positive exosomes suggests potential peripheral cholinergic dysfunction in PD, independent of the central cholinergic system. The observed alterations in AChE activity provide valuable insights into the association between cholinergic dysfunction and the pathogenesis of PD.

Structural Identification of Ginsenoside Based on UPLC-QTOF-MS of Black Ginseng (Panax Ginseng C.A. Mayer).

Black ginseng (BG) is processed ginseng traditionally made in Korea via the steaming and drying of ginseng root through three or more cycles, leading to changes in its appearance due to the Maillard reaction on its surface, resulting in a dark coloration. In this study, we explored markers for differentiating processed ginseng by analyzing the chemical characteristics of BG. We elucidated a new method for the structural identification of ginsenoside metabolites and described the features of processed ginseng using UPLC-QTOF-MS in the positive ion mode. We confirmed that maltose, glucose, and fructose, along with L-arginine, L-histidine, and L-lysine, were the key compounds responsible for the changes in the external quality of BG. These compounds can serve as important metabolic markers for distinguishing BG from conventionally processed ginseng. The major characteristics of white ginseng, red ginseng, and BG can be distinguished based on their high-polarity and low-polarity ginsenosides, and a precise method for the structural elucidation of ginsenosides in the positive ion mode is presented.

Crystal structure of Bak bound to the BH3 domain of Bnip5, a noncanonical BH3 domain-containing protein.

The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.

Association of plasma amyloid-ß oligomerization with theta/beta ratio in older adults.

Background: Oligomeric Aß (OAß) is a promising candidate marker for Alzheimer's disease (AD) diagnosis. Electroencephalography (EEG) is a potential tool for early detection of AD. Still, whether EEG power ratios, particularly the theta/alpha ratio (TAR) and theta/beta ratio (TBR), reflect Aß burden-a primary mechanism underlying cognitive impairment and AD. This study investigated the association of TAR and TBR with amyloid burden in older adults based on MDS-OAß levels. Methods: 529 individuals (aged ≥60 years) were recruited. All participants underwent EEG (MINDD SCAN, Ybrain Inc., South Korea) and AlzOn™ test (PeopleBio Inc., Gyeonggi-do, Republic of Korea) for quantifying MDS-OAß values in the plasma. EEG variables were log-transformed to normalize the data distribution. Using the MDS-OAß cutoff value (0.78 ng/mL), all participants were classified into two groups: high MDS-OAß and low MDS-OAß. Results: Participants with high MDS-OAß levels had significantly higher TARs and TBRs than those with low MDS-OAß levels. The log-transformed TBRs in the central lobe (ß = 0.161, p = 0.0026), frontal lobe (ß = 0.145, p = 0.0044), parietal lobe (ß = 0.166, p = 0.0028), occipital lobe (ß = 0.158, p = 0.0058), and temporal lobe (beta = 0.162, p = 0.0042) were significantly and positively associated with increases in MDS-OAß levels. After adjusting for the Bonferroni correction, the TBRs in all lobe regions, except the occipital lobe, were significantly associated with increased MDS-OAß levels. Conclusion: We found a significant association of MDS-OAß with TBR in older adults. This finding indicates that an increase in amyloid burden may be associated with an increase in the low-frequency band and a decrease in the relatively high-frequency band.

Insights into the recognition mechanism in the UBR box of UBR4 for its specific substrates.

The N-end rule pathway is a proteolytic system involving the destabilization of N-terminal amino acids, known as N-degrons, which are recognized by N-recognins. Dysregulation of the N-end rule pathway results in the accumulation of undesired proteins, causing various diseases. The E3 ligases of the UBR subfamily recognize and degrade N-degrons through the ubiquitin-proteasome system. Herein, we investigated UBR4, which has a distinct mechanism for recognizing type-2 N-degrons. Structural analysis revealed that the UBR box of UBR4 differs from other UBR boxes in the N-degron binding sites. It recognizes type-2 N-terminal amino acids containing an aromatic ring and type-1 N-terminal arginine through two phenylalanines on its hydrophobic surface. We also characterized the binding mechanism for the second ligand residue. This is the report on the structural basis underlying the recognition of type-2 N-degrons by the UBR box with implications for understanding the N-end rule pathway.

Cellular toxicity profile of a new ophthalmic sponge.

Introduction: Ophthalmic sponges are used for cleaning the eye surface and absorbing fluids during ophthalmic procedures. This study compared the biological safety and stability of a new ophthalmic sponge, Occucell® (OccuTech Inc, Seongnam, Korea), on the human conjunctival epithelial cells with those of preexisting products to evaluate its clinical application.Materials and Methods: The cytotoxicity of four products, Occucell, a new product, Ultracell®, Eyetec-1, and Eyetec-2, on conjunctival epithelial cells, was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) analysis. Additionally, human conjunctival epithelial cells were stained with a Live & Dead marker and observed using a fluorescence microscope. To evaluate the effect of the ophthalmic sponges on the secretion of IL-1ß and TNF-α, cultured conjunctival epithelial cells were treated with 0.5% DMSO eluates of the ophthalmic sponges, and IL-1ß and TNF-α mRNA levels were estimated using real-time polymerase chain reaction assays.Results: Cells treated with Occucell showed comparable viability to those treated with other preexisting products. Conjunctival epithelial cells showed more than 90% viability when treated with the ophthalmic sponge extracts, as determined by the MTT assay. No significant differences in the number of live & dead cells were observed between the control and treatment groups. Cells treated with all four ophthalmic sponge eluates showed similar IL-1ß and TNF-α mRNA levels.Discussion: Occucell, an eye sponge used during ophthalmic surgery in clinical practice, did not affect the viability of conjunctival epithelial cells, and more than 90% of the cells were viable after the treatment. Further, Occucell showed similar effects on IL-1ß and TNF-α secretion as that of other ophthalmic sponges used in the clinic. This suggested that Occucell is a safe product comparable to the preexisting products.

A natural variation in the RNA polymerase of severe fever with thrombocytopenia syndrome virus enhances viral replication and in vivo virulence.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease with high mortality in Eastern Asia. The disease is caused by the SFTS virus (SFTSV), also known as Dabie bandavirus, which has a segmented RNA genome consisting of L, M, and S segments. Previous studies have suggested differential viral virulence depending on the genotypes of SFTSV; however, the critical viral factor involved in the differential viral virulence is unknown. Here, we found a significant difference in viral replication in vitro and virulence in vivo between two Korean isolates belonging to the F and B genotypes, respectively. By generating viral reassortants using the two viral strains, we demonstrated that the L segment, which encodes viral RNA-dependent RNA polymerase (RdRp), is responsible for the enhanced viral replication and virulence. Comparison of amino acid sequences and viral replication rates revealed a point variation, E251K, on the surface of RdRp to be the most significant determinant for the enhanced viral replication rate and in vivo virulence. The effect of the variation was further confirmed using recombinant SFTSV generated by reverse genetic engineering. Therefore, our results indicate that natural variations affecting the viral replicase activity could significantly contribute to the viral virulence of SFTSV.

Association Between the Loss of Gait Harmony and Cognitive Impairment: Cross-Sectional Study.

BACKGROUND: Functional limitations and disabilities have been associated with a decrease in cognitive function due to increasing age. Gait performance and cognitive function have been associated with gait variability in executive function, the phase domain in memory, and gait abnormalities in cognitive decline. OBJECTIVE: Our study aimed to investigate whether gait harmony was associated with cognitive function in the older adult population. Moreover, we aimed to investigate whether gait harmony was associated with cognitive function and explore each cognitive function in a specific harmonic state. METHODS: The study population included 510 adults aged ≥60 years who visited the Department of Neurology at the Veterans Health Service Medical Center, Seoul, South Korea. Gait data were collected using a 3D motion capture device with a wireless inertial measurement unit system. For cognitive function assessments, we used the Seoul Neuropsychological Screening Battery-Core test, which evaluates the level of cognitive function or impairment in 5 cognitive domains. RESULTS: In general, the association between the Seoul Neuropsychological Screening Battery-Core tests and the stance-to-swing ratio in the >1.63 ratio group yielded lower ß coefficients than those in the 1.50-1.63 ratio group. After adjustment for confounders, the odds ratio (OR) for the Digit Symbol Coding test (adjusted OR 0.42, 95% CI 0.20-0.88) and the Korean version of the Color Word Stroop Test: 60 seconds (adjusted OR 0.51, 95% CI 0.29-0.89) for frontal and executive function were significantly lower for the >1.63 ratio group than the reference group. CONCLUSIONS: Our findings suggest that the gait phase ratio is a valuable indicator of walking deficits and may also be associated with cognitive impairment in older adults.

Brain-derived neurotrophic factor levels and macular ganglion cell-inner plexiform layer thickness in macular telangiectasia type 2.

PURPOSE: To determine brain-derived neurotrophic factor (BDNF) levels in the serum and aqueous humor (AH) and assess the relationship between BDNF levels and the thickness of the macular ganglion cell-inner plexiform layer (GCIPL) in macular telangiectasia type 2 (MacTel). METHODS: This study included 25 patients with MacTel (MacTel group) and 25 control subjects (control group). The levels of BDNF in the serum and AH were tested using an enzyme-binding immunosorbent assay. GCIPL thickness was measured by segmentation analysis using optical coherence tomography (OCT). RESULTS: There was no significant difference in the mean serum BDNF levels between the MacTel and control groups (p = 0.145). The average BDNF level in the AH was significantly lower than that in the control group (p = 0.026). OCT segmentation analyses revealed that the minimum GCIPL thickness was significantly lower in the MacTel group than in the control group (p = 0.039). In the correlation analysis of BDNF levels with GCIPL thickness, significant correlations existed between the BDNF level of the AH and minimum GCIPL thickness in the MacTel group. CONCLUSION: The concentration of BDNF in the AH was decreased in the MacTel group, and this reduction was related to the minimum GCIPL thickness. The low BDNF levels detected in the MacTel group may have resulted in thinning of the GCIPL due to the loss of retinal ganglion cells.

Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains.

BACKGROUND: Owing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems. RESULTS: Here, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains. CONCLUSION: The Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.

High-throughput analysis of amniotic fluid proteins associated with histological chorioamnionitis in preterm premature rupture of membranes using an antibody-based microarray.

PROBLEM: To identify potential proteins in the amniotic fluid (AF) that may be associated with histologic chorioamnionitis (HCA) in patients with preterm premature rupture of membranes (PPROM) using antibody-based microarray analysis. METHOD OF STUDY: This was a retrospective cohort study involving 100 singleton pregnant women with PPROM at 24-34 weeks who underwent amniocentesis and delivered within 120 h of amniocentesis. First, the AF proteomes of 15 patients with PPROM and HCA were compared with those of 15 gestational age-matched patients without HCA using a protein microarray. Next, 12 candidate proteins associated with HCA were further validated in 100 consecutive patients with PPROM by ELISA. RESULTS: Of 507 proteins assessed in the microarray analysis, 46 showed significant intergroup differences. Further quantification confirmed that the levels of EN-RAGE, IL-6, MMP-9, TNFR2, SPARC, TSP2, and uPA were higher in the AF of PPROM patients with HCA than in those without. Multivariate analyses also showed that elevated AF EN-RAGE, IL-6, MMP-9, and TNFR2 levels were independently associated with HCA when adjusted for baseline variables. The frequency of the highest quartile of the aforementioned proteins significantly increased as the total grade of HCA increased; the risk of HCA significantly increased with increasing AF levels of each protein (P for trend < .001). CONCLUSIONS: Using protein-antibody microarray technology, we discovered several potential AF proteins (EN-RAGE, IL-6, MMP-9, and TNFR2) independently associated with HCA in patients with PPROM. Furthermore, we demonstrated a direct correlation between the gradation of the intra-amniotic inflammatory response and HCA severity.

Recapitulated Crosstalk between Cerebral Metastatic Lung Cancer Cells and Brain Perivascular Tumor Microenvironment in a Microfluidic Co-Culture Chip.

Non-small cell lung carcinoma (NSCLC), which affects the brain, is fatal and resistant to anti-cancer therapies. Despite innate, distinct characteristics of the brain from other organs, the underlying delicate crosstalk between brain metastatic NSCLC (BM-NSCLC) cells and brain tumor microenvironment (bTME) associated with tumor evolution remains elusive. Here, a novel 3D microfluidic tri-culture platform is proposed for recapitulating positive feedback from BM-NSCLC and astrocytes and brain-specific endothelial cells, two major players in bTME. Advanced imaging and quantitative functional assessment of the 3D tri-culture model enable real-time live imaging of cell viability and separate analyses of genomic/molecular/secretome from each subset. Susceptibility of multiple patient-derived BM-NSCLCs to representative targeted agents is altered and secretion of serpin E1, interleukin-8, and secreted phosphoprotein 1, which are associated with tumor aggressiveness and poor clinical outcome, is increased in tri-culture. Notably, multiple signaling pathways involved in inflammatory responses, nuclear factor kappa-light-chain-enhancer of activated B cells, and cancer metastasis are activated in BM-NSCLC through interaction with two bTME cell types. This novel platform offers a tool to elucidate potential molecular targets and for effective anti-cancer therapy targeting the crosstalk between metastatic cancer cells and adjacent components of bTME.

Cell Factory Engineering of Undomesticated Bacillus Strains Using a Modified Integrative and Conjugative Element for Efficient Plasmid Delivery.

A large number of Bacillus strains have been isolated from various environments and many of them have great potential as cell factories. However, they have been rarely developed as cell factories due to their poor transformation efficiency. In this study, we developed a highly efficient plasmid delivery system for undomesticated Bacillus strains using a modified integrative and conjugative element (MICE), which was designed to be activated by an inducer, prevent self-transfer, and deliver desired plasmids to the recipient cells. The MICE system was demonstrated to successfully introduce a gfp-containing plasmid into all 41 undomesticated Bacillus subtilis strains tested and eight other Bacillus species. The MICE was used to deliver a cytosine base editor (CBE)-based multiplex genome-editing tool for the cell factory engineering of the Bacillus species. The introduced CBE enabled one-step inactivation of the major extracellular protease genes of the tested strains. The engineered strains were used as hosts for heterologous expression of nattokinase, which resulted in various enzyme expression levels. The results suggested that the MICE and CBE systems can be powerful tools for genetic engineering of undomesticated Bacillus strains, and greatly contribute to the expansion of the Bacillus cell factory.