Engineering Escherichia coli for constitutive production of monophosphoryl lipid A vaccine adjuvant.

During the COVID-19 pandemic, expedient vaccine production has been slowed by the shortage of safe and effective raw materials, such as adjuvants, essential components to enhance the efficacy of vaccines. Monophosphoryl lipid A (MPLA) is a potent and safe adjuvant used in human vaccines, including the Shingles vaccine, Shingrix. 3-O-desacyl-4'-monophosphoryl lipid A (MPL), a representative MPLA adjuvant commercialized by GSK, was prepared via chemical conversion of precursors isolated from Salmonella typhimurium R595. However, the high price of these materials limits their use in premium vaccines. To combat the scarcity and high cost of safe raw materials for vaccines, we need to develop a feasible MPLA production method that is easily scaled up to meet industrial requirements. In this study, we engineered peptidoglycan and outer membrane biosynthetic pathways in Escherichia coli and developed a Escherichia coli strain, KHSC0055, that constitutively produces EcML (E. coli-produced monophosphoryl lipid A) without additives such as antibiotics or overexpression inducers. EcML production was optimized on an industrial scale via high-density fed-batch fermentation, and obtained 2.7 g of EcML (about 135,000 doses of vaccine) from a 30-L-scale fermentation. Using KHSC0055, we simplified the production process and decreased the production costs of MPLA. Then, we applied EcML purified from KHSC0055 as an adjuvant for a COVID-19 vaccine candidate (EuCorVac-19) currently in clinical trial stage III in the Philippines. By probing the efficacy and safety of EcML in humans, we established KHSC0055 as an efficient cell factory for MPLA adjuvant production.

Quorum Quenching in Membrane Bioreactors for Fouling Retardation: Complexity Provides Opportunities.

The occurrence of biofouling restricts the widespread application of membrane bioreactors (MBRs) in wastewater treatment. Regulation of quorum sensing (QS) is a promising approach to control biofouling in MBRs, yet the underlying mechanisms are complex and remain to be illustrated. A fundamental understanding of the relationship between QS and membrane biofouling in MBRs is lacking, which hampers the development and application of quorum quenching (QQ) techniques in MBRs (QQMBRs). While many QQ microorganisms have been isolated thus far, critical criteria for selecting desirable QQ microorganisms are still missing. Furthermore, there are inconsistent results regarding the QQ lifecycle and the effects of QQ on the physicochemical characteristics and microbial communities of the mixed liquor and biofouling assemblages in QQMBRs, which might result in unreliable and inefficient QQ applications. This review aims to comprehensively summarize timely QQ research and highlight the important yet often ignored perspectives of QQ for biofouling control in MBRs. We consider what this "information" can and cannot tell us and explore its values in addressing specific and important questions in QQMBRs. Herein, we first examine current analytical methods of QS signals and discuss the critical roles of QS in fouling-forming microorganisms in MBRs, which are the cornerstones for the development of QQ technologies. To achieve targeting QQ strategies in MBRs, we propose the substrate specificity and degradation capability of isolated QQ microorganisms and the surface area and pore structures of QQ media as the critical criteria to select desirable functional microbes and media, respectively. To validate the biofouling retardation efficiency, we further specify the QQ effects on the physicochemical properties, microbial community composition, and succession of mixed liquor and biofouling assemblages in MBRs. Finally, we provide scale-up considerations of QQMBRs in terms of the debated QQ lifecycle, practical synergistic strategies, and the potential cost savings of MBRs. This review presents the limitations of classic QS/QQ hypotheses in MBRs, advances the understanding of the role of QS/QQ in biofouling development/retardation in MBRs, and builds a bridge between the fundamental understandings and practical applications of QQ technology.

Innovative Biofouling Control for Membrane Bioreactors in Cold Regions by Inducing Environmental Adaptation in Quorum-Quenching Bacteria.

Bacterial quorum quenching (QQ), whose mechanism involves the degradation of quorum-sensing signal molecules, is an effective strategy for controlling biofouling in membrane bioreactors (MBRs). However, MBRs operated at low temperatures, either due to cold climates or seasonal variations, exhibit severe deterioration in QQ efficiency. In this study, a modified culture method for Rhodococcus sp. BH4, a QQ bacterium, was developed to induce environmental adaptation in cold regions. BH4-L, which was prepared by the modified culture method, showed enhancement in QQ efficiency at low temperatures. The higher QQ efficiency obtained by employing BH4-L at 10 °C (compared with that obtained by employing BH4 at 10 °C) was attributed to the higher live/dead cell ratio in the BH4-L-entrapping beads. When BH4-L-entrapping beads were applied to lab-scale MBRs operated at low temperatures, membrane biofouling in MBRs at low temperatures was successfully mitigated because BH4-L could substantially reduce the concentration of signal molecules (N-acyl homoserine lactones) in the biocake. Employing BH4-L in QQ-MBRs could offer a novel solution to the problem of severe membrane biofouling in MBRs in cold regions.

Loss of splicing factor IK impairs normal skeletal muscle development.

BACKGROUND: IK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo. RESULTS: The ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells. CONCLUSION: This study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

A Fluorescence-Polarization-Based Lipopolysaccharide-Caspase-4 Interaction Assay for the Development of Inhibitors.

Recognition of intracellular lipopolysaccharide (LPS) by Caspase-4 (Casp-4) is critical for host defense against Gram-negative pathogens. LPS binds to the N-terminal caspase activation and recruitment domain (CARD) of procaspase-4, leading to auto-proteolytic activation followed by pro-inflammatory cytokine release and pyroptotic cell death. Aberrant hyper-activation of Casp-4 leads to amplification of the inflammatory response linked to sepsis. While the active site of a caspase has been targeted with peptide inhibitors, inhibition of LPS-Casp-4 interaction is an emerging strategy for the development of selective inhibitors with a new mode of action for treating infectious diseases and sepsis induced by LPS. In this study, a high-throughput screening (HTS) system based on fluorescence polarization (FP) was devised to identify inhibitors of the LPS and Casp-4 interaction. Using HTS and IC50 determination and subsequently showing inhibited Casp-4 activity, we demonstrated that the LPS-Casp-4 interaction is a druggable target for Casp-4 inhibition and possibly a non-canonical inflammatory pathway.

Altrenogest affects expression of galectin-3 and fibroblast growth factor 9 in the reproductive tract of sows.

A synthetic progestin altrenogest (ALT) is used to synchronize the estrus cycle of swine for fixed-time artificial insemination (AI) and has been shown to improve follicular development and reproductive performances in post-weaning sows. However, the effects of ALT treatment on reproductive tracts, including the ovaries, oviducts and uterus have not been yet clarified. In this study, we examined the expression of genes involved in endometrial responses in ALT-treated sows. ALT did not significantly alter luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol profiles in blood compared to untreated control. Quantitative RT-polymerase chain reaction (qRT-PCR) analysis showed that the expression of genes encoding galectin-3 (LGALS3) and fibroblast growth factor 9 (FGF9) was upregulated in the reproductive tracts of ALT-treated sows, including the ovaries, oviducts and uteri. Moreover, ALT treatment induced the expression of FGF9 and galectin-3 proteins, and promoted their localization to the luminal epithelium of the oviducts and uterus. Our findings suggest that the enhancement of reproductive performance shown by ALT-treated sows is associated with the upregulation of galectin-3 and FGF9, which are essential for endometrial receptivity, successful implantation, and pregnancy.

Zinc-dependent activation of the Pho8 alkaline phosphatase in Schizosaccharomyces pombe.

Genome-wide analyses have revealed that during metal ion starvation, many cells undergo programmed changes in their transcriptome or proteome that lower the levels of abundant metalloproteins, conserving metal ions for more critical functions. Here we investigated how changes in cellular zinc status affect the expression and activity of the zinc-requiring Pho8 alkaline phosphatase from fission yeast (Schizosaccharomyces pombe). In S. pombe, Pho8 is a membrane-tethered and processed glycoprotein that resides in the vacuole. Using alkaline phosphatase activity assays along with various biochemical analyses, we found that Pho8 is active when zinc is plentiful and inactive when zinc is limited. Although Pho8 activity depended on zinc, we also found that higher levels of pho8 mRNAs and Pho8 protein accumulate in zinc-deficient cells. To gain a better understanding of the inverse relationship between pho8 mRNA levels and Pho8 activity, we examined the effects of zinc on the stability and processing of the Pho8 protein. We show that Pho8 is processed regardless of zinc status and that mature Pho8 accumulates under all conditions. We also noted that alkaline phosphatase activity is rapidly restored when zinc is resupplied to cells, even in the presence of the protein synthesis inhibitor cycloheximide. Our results suggest that S. pombe cells maintain inactive pools of Pho8 proteins under low-zinc conditions and that these pools facilitate rapid restoration of Pho8 activity when zinc ions become available.

Extracellular pyruvate kinase M2 facilitates cell migration by upregulating claudin-1 expression in colon cancer cells.

Extensive studies have been reported the non-canonical functions of pyruvate kinase M2 (PKM2) as a kinase, transcriptional regulator, and even cell-to-cell communicator, emphasizing its importance in various signaling pathways. However, the role of secreted PKM2 in cancer progression and its signaling pathway is yet to be elucidated. In this study, we found that extracellular PKM2 enhanced the migration of low-metastatic, benign colon cancer cells by upregulating claudin-1 expression and internalizing it to the cytoplasm and nucleus. Knock-down of claudin-1 significantly reduced extracellular PKM2-induced cell migration. Inhibition of either protein kinase C (PKC) or epidermal growth factor receptor (EGFR) resulted in a reduction of extracellular PKM2-mediated claudin-1 expression, suggesting EGFR-PKC-claudin-1 as a signaling pathway in the extracellular PKM2-mediated tumorigenesis of colon cancer cells.

Metabolic engineering of Escherichia coli to produce a monophosphoryl lipid A adjuvant.

Monophosphoryl lipid A (MPLA) species, including MPL (a trade name of GlaxoSmithKline) and GLA (a trade name of Immune Design, a subsidiary of Merck), are widely used as an adjuvant in vaccines, allergy drugs, and immunotherapy to boost the immune response. Even though MPLA is a derivative of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, bacterial strains producing MPLA have not been found in nature nor engineered. In fact, MPLA generation involves expensive and laborious procedures based on synthetic routes or chemical transformation of precursors isolated from Gram-negative bacteria. Here, we report the engineering of an Escherichia coli strain for in situ production and accumulation of MPLA. Furthermore, we establish a succinct method for purifying MPLA from the engineered E. coli strain. We show that the purified MPLA (named EcML) stimulates the mouse immune system to generate antigen-specific IgG antibodies similarly to commercially available MPLA, but with a dramatically reduced manufacturing time and cost. Our system, employing the first engineered E. coli strain that directly produces the adjuvant EcML, could transform the current standard of industrial MPLA production.

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4).

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O2 -based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm2 membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 µm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m-2 ·d-1 maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.

Effects of Quorum Quenching on Biofilm Metacommunity in a Membrane Bioreactor.

Quorum quenching (QQ) has received attention for the control of biofilms, e.g., biofilms that cause biofouling in membrane bioreactors (MBRs). Despite the efficacy of QQ on biofouling, it is elusive how QQ influences biofilm formation on membranes. A pilot-scale QQ-MBR and non-QQ-MBR were identically operated for 4 days and 8 days to destructively sample the membranes. QQ prolonged the membrane filterability by 43% with no harmful influence on MBR performance. qPCR showed no effect of QQ on microbial density during either of these time periods. Community comparisons revealed that QQ influenced the bacterial and fungal community structures, and the fungal structure corresponded with the bacterial structure. Metacommunity and spatial analyses showed that QQ induced structural variation rather than compositional variation of bacteria and fungi. Moreover, QQ considerably enhanced the bacterial dispersal across membrane during the early development. As the dispersal enhancement by QQ counteracted the ecological drift, it eliminated the distance-decay relationship, reflecting a neutral theory archetype of metacommunity. Network analyses showed that QQ substantially reduced the amount and magnitude of interactions, e.g., competition and cooperation, for bacteria and fungi, and weakened their network structures, irrespective of time. Additionally, QQ suppressed the growth of specific microbial species (e.g., Acinetobacter), abundant and widespread at the early stage. These findings suggest that QQ influenced the community dynamics at the regional and local levels, correspondingly the ecological selection and dispersal processes, during the biofilm development.

Preparation of a mesoporous silica quorum quenching medium for wastewater treatment using a membrane bioreactor.

Various quorum quenching (QQ) media have been developed to mitigate membrane biofouling in a membrane bioreactor (MBR). However, most are expensive, unstable and easily trapped in hollow fibre membranes. Here, a sol-gel method was used to develop a mesoporous silica medium entrapping a QQ bacterial strain (Rhodococcus sp. BH4). The new silica QQ medium was able to remove quorum sensing signalling molecules via both adsorption (owing to their mesoporous hydrophobic structure) and decomposition with an enzyme (lactonase), preventing MBR biofouling without affecting the water quality. It also demonstrated a relatively long life span due to its non-biodegradability and its relatively small particle size (<1.0 mm), which makes it less likely to clog in a hollow fibre membrane module.

The expression and localization of V-ATPase and cytokeratin 5 during postnatal development of the pig epididymis.

OBJECTIVE: We examined the localization and expression of H+ pumping vacuolar ATPase (V-ATPase) and cytokeratin 5 (KRT5) in the epididymis of pigs, expressed in clear and basal cells, respectively, during postnatal development. METHODS: Epididymides were obtained from pigs at 1, 7, 21, 60, 120, and 180 days of age; we observed the localization and expression patterns of V-ATPase and KRT5 in the different regions of these organs, namely, the caput, corpus, and cauda. The differentiation of epididymal epithelial cells was determined by immunofluorescence labeling using cell-type-specific markers and observed using confocal microscopy. RESULTS: At postnatal day 5 (PND5), the localization of clear cells commenced migration from the cauda toward the caput. Although at PND120, goblet-shaped clear cells were detected along the entire length of the epididymis, those labeled for V-ATPase had disappeared from the corpus to cauda and were maintained only in the caput epididymis in adult pigs. In contrast, whereas basal cells labeled for KRT5 were only present in the vas deferens at birth, they were detected in all regions of the epididymis at PND60. These cells were localized at the base of the epithelium; however, no basal cells characterized by luminally extending cell projections were observed in any of the adult epididymides examined. CONCLUSION: The differentiation of clear and basal cells progressively initiates in a retrograde manner from the cauda to the caput epididymis. The cell-type-specific distribution and localization of the epithelial cells play important roles in establishing a unique luminal environment for sperm maturation and storage in the pig epididymis.

Quorum sensing: an emerging link between temperature and membrane biofouling in membrane bioreactors.

Lab-scale membrane bioreactors (MBRs) were investigated at 12, 18, and 25 °C to identify the correlation between quorum sensing (QS) and biofouling at different temperatures. The lower the reactor temperature, the more severe the membrane biofouling measured in terms of the transmembrane pressure (TMP) during filtration. More extracellular polymeric substances (EPSs) that cause biofouling were produced at 18 °C than at 25 °C, particularly polysaccharides, closely associated with QS via the production of N-acyl homoserine lactone (AHL). However, at 12 °C, AHL production decreased, but the release of EPSs due to deflocculation increased the soluble EPS concentration. To confirm the temperature effect related to QS, bacteria producing AHL were isolated from MBR sludge and identified as Aeromonas sp., Leclercia sp., and Enterobacter sp. through a 16S rDNA sequencing analysis. Batch assays at 18 and 25 °C showed that there was a positive correlation between QS through AHL and biofilm formation in that temperature range.

Production of human tissue-type plasminogen activator (htPA) using in vitro cultured transgenic pig mammary gland cells.

Tissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production.

Stopping Autoinducer-2 Chatter by Means of an Indigenous Bacterium ( Acinetobacter sp. DKY-1): A New Antibiofouling Strategy in a Membrane Bioreactor for Wastewater Treatment.

Bacterial quorum quenching (QQ) by means of degrading signaling molecules has been applied to antibiofouling strategies in a membrane bioreactor (MBR) for wastewater treatment. However, the target signaling molecules have been limited to N-acyl homoserine lactones participating in intraspecies quorum sensing. Here, an approach to disrupting autoinducer-2 (AI-2) signaling molecules participating in interspecies quorum sensing was pursued as a next-generation antibiofouling strategy in an MBR for wastewater treatment. We isolated an indigenous QQ bacterium ( Acinetobacter sp. DKY-1) that can attenuate the expression of the quorum-sensing (QS) response through the inactivation of an autoinducer-2 signaling molecule, 4,5-dihydroxy-2,3-pentanedione (DPD), among four kinds of autoinducer-2 QS bacteria. DKY-1 released AI-2 QQ compounds, which were verified to be hydrophilic with a molecular weight of <400 Da. The addition of DKY-1 entrapping beads into an MBR significantly decreased DPD concentration and remarkably reduced membrane biofouling. This new approach, combining molecular biology with wastewater engineering, could enlarge the range of QQ-MBR for antibiofouling and energy savings in the field of wastewater treatment.

NANOG gene expression is regulated by the ETS transcription factor ETV4 in human embryonic carcinoma NCCIT cells.

We demonstrated that ETV4 is a transcriptional activator of the NANOG gene in human embryonic carcinoma NCCIT cells. The endogenous expression of NANOG and ETV4 in naïve cells was significantly down-regulated upon differentiation and by shRNA-mediated knockdown of ETV4. NANOG transcription was significantly upregulated by ETV4 overexpression. A putative ETS binding site (EBS) is present in the region (-285 to -138) of the proximal promoter. Site-directed mutagenesis of the putative EBS (-196AGGATT-191) abolished NANOG promoter activity and ETV4 interacted with this putative EBS both in vivo and in vitro. Our data provide the molecular details of ETV4-mediated NANOG gene expression.

Esculetin exerts anti-proliferative effects against non-small-cell lung carcinoma by suppressing specificity protein 1 in vitro.

Esculetin, a coumarin derivative, is a phenolic compound isolated from Artemisia capillaris, Citrus limonia, and Euphorbia lathyris. Although it has been reported to have anti-inflammatory, anti-oxidant, and anti-proliferative activities in several human cancers, its anti-proliferative activity against non-small-cell lung carcinoma (NSCLC) and the molecular mechanisms involved have not been adequately elucidated. In this study, we used two NSCLC cell lines (NCI-H358 and NCI-H1299) to investigate the anti-proliferative activity and apoptotic effect of esculetin. Our data showed that esculetin-treated cells exhibited reduced proliferation and apoptotic cell morphologies. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly suppressed by esculetin in a dose- and time-dependent manner. Furthermore, the levels of p27 and p21, two key regulators of the cell cycle, were up-regulated by the esculetin-mediated down-regulation of Sp1; the level of a third cell-cycle regulator, survivin, was decreased, resulting in caspase-dependent apoptosis. Therefore, we conclude that esculetin could be a potent anti-proliferative agent in patients with NSCLC.

Crystal structure and activity of Francisella novicida UDP-N-acetylglucosamine acyltransferase.

The first step of lipid A biosynthesis in Escherichia coli (E. coli) is catalyzed by LpxA (EcLpxA), an acyltransferase selective for UDP-N-acetylglucosamine (UDP-GlcNAc) and R-3-hydroxymyristoyl-acyl carrier protein (3-OH-C14-ACP), and is an essential step in majority of Gram-negative bacteria. Since the majority of lipid A species isolated from F. novicida contains 3-OH-C16 or 3-OH-C18 at its C3 and C3' positions, FnLpxA was thought to be selective for longer acyl chain (3-OH-C16 and 3-OH-C18) over short acyl chain (3-OH-C14, 3-OH-C12, and 3-OH-C10). Here we demonstrate that Francisella novicida (F. novicida) lpxA functionally complements an E. coli lpxA knockout mutant and efficiently transfers 3-OH-C14 as well as 3-OH-C16 in E. coli. Our results implicate that the acyl chain length of lipid A is determined by several factors including acyl chain selectivity of LpxA and downstream enzymes, as well as the composition of the acyl-ACP pool in vivo. We also report the crystal structure of F. novicida LpxA (FnLpxA) at 2.06 Å. The N-terminal parallel beta-helix (LßH) and C-terminal alpha-helical domain are similar to other reported structures of LpxAs. However, our structure indicates that the supposed ruler residues for hydrocarbon length, 171L in one monomer and 168H in the adjacent monomer in a functional trimer of FnLpxA, are located just 3.8 Å apart that renders not enough space for binding of 3-OH-C12 or longer acyl chains. This implicates that FnLpxA may have an alternative hydrophobic pocket, or the acyl chain may bend while binding to FnLpxA. In addition, the FnLpxA structure suggests a potential inhibitor binding site for development of antibiotics.