All-in-one IQ toggle switches with high versatilities for fine-tuning of transgene expression in mammalian cells and tissues.

The transgene toggling device is recognized as a powerful tool for gene- and cell-based biological research and precision medicine. However, many of these devices often operate in binary mode, exhibit unacceptable leakiness, suffer from transgene silencing, show cytotoxicity, and have low potency. Here, we present a novel transgene switch, SIQ, wherein all the elements for gene toggling are packed into a single vector. SIQ has superior potency in inducing transgene expression in response to tebufenozide compared with the Gal4/UAS system, while completely avoiding transgene leakiness. Additionally, the ease and versatility of SIQ make it possible with a single construct to perform transient transfection, establish stable cell lines by targeting a predetermined genomic locus, and simultaneously produce adenovirus for transduction into cells and mammalian tissues. Furthermore, we integrated a cumate switch into SIQ, called SIQmate, to operate a Boolean AND logic gate, enabling swift toggling-off of the transgene after the removal of chemical inducers, tebufenozide and cumate. Both SIQ and SIQmate offer precise transgene toggling, making them adjustable for various researches, including synthetic biology, genome engineering, and therapeutics.

AAV-compatible optogenetic tools for activating endogenous calcium channels in vivo.

Calcium ions (Ca2+) play pivotal roles in regulating diverse brain functions, including cognition, emotion, locomotion, and learning and memory. These functions are intricately regulated by a variety of Ca2+-dependent cellular processes, encompassing synaptic plasticity, neuro/gliotransmitter release, and gene expression. In our previous work, we developed 'monster OptoSTIM1' (monSTIM1), an improved OptoSTIM1 that selectively activates Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane through blue light, allowing precise control over intracellular Ca2+ signaling and specific brain functions. However, the large size of the coding sequence of monSTIM1 poses a limitation for its widespread use, as it exceeds the packaging capacity of adeno-associated virus (AAV). To address this constraint, we have introduced monSTIM1 variants with reduced coding sequence sizes and established AAV-based systems for expressing them in neurons and glial cells in the mouse brain. Upon expression by AAVs, these monSTIM1 variants significantly increased the expression levels of cFos in neurons and astrocytes in the hippocampal CA1 region following non-invasive light illumination. The use of monSTIM1 variants offers a promising avenue for investigating the spatiotemporal roles of Ca2+-mediated cellular activities in various brain functions. Furthermore, this toolkit holds potential as a therapeutic strategy for addressing brain disorders associated with aberrant Ca2+ signaling.

TDAG51 promotes transcription factor FoxO1 activity during LPS-induced inflammatory responses.

Tight regulation of Toll-like receptor (TLR)-mediated inflammatory responses is important for innate immunity. Here, we show that T-cell death-associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the transcription factor FoxO1, regulating inflammatory mediator production in the lipopolysaccharide (LPS)-induced inflammatory response. TDAG51 induction by LPS stimulation was mediated by the TLR2/4 signaling pathway in bone marrow-derived macrophages (BMMs). LPS-induced inflammatory mediator production was significantly decreased in TDAG51-deficient BMMs. In TDAG51-deficient mice, LPS- or pathogenic Escherichia coli infection-induced lethal shock was reduced by decreasing serum proinflammatory cytokine levels. The recruitment of 14-3-3ζ to FoxO1 was competitively inhibited by the TDAG51-FoxO1 interaction, leading to blockade of FoxO1 cytoplasmic translocation and thereby strengthening FoxO1 nuclear accumulation. TDAG51/FoxO1 double-deficient BMMs showed significantly reduced inflammatory mediator production compared with TDAG51- or FoxO1-deficient BMMs. TDAG51/FoxO1 double deficiency protected mice against LPS- or pathogenic E. coli infection-induced lethal shock by weakening the systemic inflammatory response. Thus, these results indicate that TDAG51 acts as a regulator of the transcription factor FoxO1, leading to strengthened FoxO1 activity in the LPS-induced inflammatory response.

Exploitation of a novel adjuvant for polymyxin B against multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Although polymyxin has been used as a last-resort antibiotic against resistant bacteria, its use is restricted due to nephrotoxicity and neurotoxicity. While the present antibiotic resistance issue compels clinicians to reconsider polymyxin use in severe illness cases, polymyxin-resistant microorganisms exert an effect. OBJECTIVES: To address the issue of antibiotic resistance, the cycle of developing new antibiotics to counteract emerging resistance must be discontinued. Here we tried to develop novel therapies that do not rely on direct antimicrobial activity and thus do not promote antibiotic resistance. METHODS: By a high-throughout screening system based on bacterial respiration, chemical compounds accelerating the antimicrobial effects of polymyxin B were screened. In vitro and in vivo tests were performed to validate adjuvanticity. In addition, membrane depolarization and total transcriptome analysis were used to determine molecular mechanisms. RESULTS: PA108, a newly discovered chemical compound, was used to eradicate polymyxin-resistant A. baumannii and three other species in the presence of polymyxin B at concentrations less than the MIC. Since this molecule lacks self-bactericidal action, we hypothesized that PA108 acts as an antibiotic adjuvant, enhancing the antimicrobial activity of polymyxin B against resistant bacteria. At working concentrations, no toxicity was observed in cell lines or mice, although co-treatment with PA108 and polymyxin B increased survival of infected mouse and decreased bacterial loads in organs. CONCLUSIONS: Boosting antibiotic efficiency through the use of antibiotic adjuvants holds significant promise for tackling the rise in bacterial antibiotic resistance.

TDAG51 deficiency attenuates dextran sulfate sodium-induced colitis in mice.

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a group of chronic inflammatory diseases of the gastrointestinal tract. Although the multifactorial etiology of IBD pathogenesis is relatively well documented, the regulatory factors that confer a risk of IBD pathogenesis remain less explored. In this study, we report that T-cell death-associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the development of dextran sulfate sodium (DSS)-induced colitis in mice. TDAG51 expression was elevated in the colon tissues of DSS-induced experimental colitis mice. TDAG51 deficiency protected mice against acute DSS-induced lethality and body weight changes and disease severity. DSS-induced structural damage and mucus secretion in colon tissues were significantly reduced in TDAG51-deficient mice compared with wild-type mice. We observed similar results in a DSS-induced chronic colitis mouse model. Finally, we showed that the production of inflammatory mediators, including proinflammatory enzymes, molecules and cytokines, was decreased in DSS-treated TDAG51-deficient mice compared with DSS-treated wild-type mice. Thus, we demonstrated that TDAG51 deficiency plays a protective role against DSS-induced colitis by decreasing the production of inflammatory mediators in mice. These findings suggest that TDAG51 is a novel regulator of the development of DSS-induced colitis and is a potential therapeutic target for IBD.

1,3-Dibenzyl-5-Fluorouracil Prevents Ovariectomy-Induced Bone Loss by Suppressing Osteoclast Differentiation.

Osteoclasts (OCs) are clinically important cells that resorb bone matrix. Accelerated bone destruction by OCs is closely linked to the development of metabolic bone diseases. In this study, we screened novel chemical inhibitors targeting OC differentiation to identify drug candidates for metabolic bone diseases. We identified that 1,3-dibenzyl-5-fluorouracil, also named OCI-101, is a novel inhibitor of osteoclastogenesis. The formation of multinucleated OCs is reduced by treatment with OCI-101 in a dose-dependent manner. OCI-101 inhibited the expression of OC markers via downregulation of receptor activator of NF-κB ligand and M-CSF signaling pathways. Finally, we showed that OCI-101 prevents ovariectomy-induced bone loss by suppressing OC differentiation in mice. Hence, these results demonstrated that OCI-101 is a good drug candidate for treating metabolic bone diseases.

Bisphenol-A impairs synaptic formation and function by RGS4-mediated regulation of BDNF signaling in the cerebral cortex.

Bisphenol-A (BPA) is a representative endocrine disruptor, widely used in a variety of products including plastics, medical equipment and receipts. Hence, most people are exposed to BPA via the skin, digestive system or inhalation in everyday life. Furthermore, BPA crosses the blood-brain barrier and is linked to multiple neurological dysfunctions found in neurodegenerative and neuropsychological disorders. However, the mechanisms underlying BPA-associated neurological dysfunctions remain poorly understood. Here, we report that BPA exposure alters synapse morphology and function in the cerebral cortex. Cortical pyramidal neurons treated with BPA showed reduced size and number of dendrites and spines. The density of excitatory synapses was also decreased by BPA treatment. More importantly, we found that BPA disrupted normal synaptic transmission and cognitive behavior. RGS4 and its downstream BDNF/NTRK2 pathway appeared to mediate the effect of BPA on synaptic and neurological function. Our findings provide molecular mechanistic insights into anatomical and physiological neurotoxic consequences related to a potent endocrine modifier.

Butylparaben Induces the Neuronal Death Through the ER Stress-Mediated Apoptosis of Primary Cortical Neurons.

Butylparaben is an organic compound that is used as an antimicrobial preservative in cosmetics and can cause neurotoxicity. However, whether butylparaben induces neuronal death is unclear. In this study, we report that butylparaben exposure induced neuronal apoptosis mediated by ER stress in primary cortical neurons. We found that butylparaben significantly inhibited the viability of primary cortical neurons and led to lactate dehydrogenase (LDH) release from primary cortical neurons. Upon exposure to butylparaben, primary cortical neurons exhibited increased levels of apoptosis-related proteins such as Cleaved-caspase3 and Bax. Interestingly, butylparaben-induced activation of apoptosis involved the upstream activation of ER stress proteins such as GRP78, CHOP, and ATF4. However, pharmacological inhibition of ER stress prevented the butylparaben-induced induction of apoptosis. Taken together, our findings suggest that butylparaben exposure activates the ER stress-mediated apoptosis of primary cortical neurons, which is closely linked with neurodegeneration in the brain. Therefore, targeting ER stress may be considered a strategy for the treatment of butylparaben-induced neurodegeneration.

Maternal Bisphenol A (BPA) Exposure Alters Cerebral Cortical Morphogenesis and Synaptic Function in Mice.

Early-life exposure to bisphenol A (BPA), synthetic compound used in polycarbonate plastic, is associated with altered cognitive and emotional behavior later in life. However, the brain mechanism underlying the behavioral deficits is unknown. Here, we show that maternal BPA exposure disrupted self-renewal and differentiation of neural progenitors during cortical development. The BPA exposure reduced the neuron number, whereas it increased glial cells in the cerebral cortex. Also, synaptic formation and transmission in the cerebral cortex were suppressed after maternal BPA exposure. These changes appeared to be associated with autophagy as a gene ontology analysis of RNA-seq identified an autophagy domain in the BPA condition. Mouse behavioral tests revealed that maternal BPA caused hyperactivity and social deficits in adult offspring. Together, these results suggest that maternal BPA exposure leads to abnormal cortical architecture and function likely by activating autophagy.

Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts.

Selenoproteins containing selenium in the form of selenocysteine are critical for bone remodeling. However, their underlying mechanism of action is not fully understood. Herein, we report the identification of selenoprotein W (SELENOW) through large-scale mRNA profiling of receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation, as a protein that is downregulated via RANKL/RANK/tumour necrosis factor receptor-associated factor 6/p38 signaling. RNA-sequencing analysis revealed that SELENOW regulates osteoclastogenic genes. SELENOW overexpression enhances osteoclastogenesis in vitro via nuclear translocation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 mediated by 14-3-3γ, whereas its deficiency suppresses osteoclast formation. SELENOW-deficient and SELENOW-overexpressing mice exhibit high bone mass phenotype and osteoporosis, respectively. Ectopic SELENOW expression stimulates cell-cell fusion critical for osteoclast maturation as well as bone resorption. Thus, RANKL-dependent repression of SELENOW regulates osteoclast differentiation and blocks osteoporosis caused by overactive osteoclasts. These findings demonstrate a biological link between selenium and bone metabolism.

Regulation of Osteoblast Differentiation by Cytokine Networks.

Osteoblasts, which are bone-forming cells, play pivotal roles in bone modeling and remodeling. Osteoblast differentiation, also known as osteoblastogenesis, is orchestrated by transcription factors, such as runt-related transcription factor 1/2, osterix, activating transcription factor 4, special AT-rich sequence-binding protein 2 and activator protein-1. Osteoblastogenesis is regulated by a network of cytokines under physiological and pathophysiological conditions. Osteoblastogenic cytokines, such as interleukin-10 (IL-10), IL-11, IL-18, interferon-γ (IFN-γ), cardiotrophin-1 and oncostatin M, promote osteoblastogenesis, whereas anti-osteoblastogenic cytokines, such as tumor necrosis factor-α (TNF-α), TNF-ß, IL-1α, IL-4, IL-7, IL-12, IL-13, IL-23, IFN-α, IFN-ß, leukemia inhibitory factor, cardiotrophin-like cytokine, and ciliary neurotrophic factor, downregulate osteoblastogenesis. Although there are gaps in the body of knowledge regarding the interplay of cytokine networks in osteoblastogenesis, cytokines appear to be potential therapeutic targets in bone-related diseases. Thus, in this study, we review and discuss our osteoblast, osteoblast differentiation, osteoblastogenesis, cytokines, signaling pathway of cytokine networks in osteoblastogenesis.

Pax5 Negatively Regulates Osteoclastogenesis through Downregulation of Blimp1.

Paired box protein 5 (Pax5) is a crucial transcription factor responsible for B-cell lineage specification and commitment. In this study, we identified a negative role of Pax5 in osteoclastogenesis. The expression of Pax5 was time-dependently downregulated by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) stimulation in osteoclastogenesis. Osteoclast (OC) differentiation and bone resorption were inhibited (68.9% and 48% reductions, respectively) by forced expression of Pax5 in OC lineage cells. Pax5 led to the induction of antiosteoclastogenic factors through downregulation of B lymphocyte-induced maturation protein 1 (Blimp1). To examine the negative role of Pax5 in vivo, we generated Pax5 transgenic (Pax5Tg) mice expressing the human Pax5 transgene under the control of the tartrate-resistant acid phosphatase (TRAP) promoter, which is expressed mainly in OC lineage cells. OC differentiation and bone resorption were inhibited (54.2-76.9% and 24.0-26.2% reductions, respectively) in Pax5Tg mice, thereby contributing to the osteopetrotic-like bone phenotype characterized by increased bone mineral density (13.0-13.6% higher), trabecular bone volume fraction (32.5-38.1% higher), trabecular thickness (8.4-9.0% higher), and trabecular number (25.5-26.7% higher) and decreased trabecular spacing (9.3-10.4% lower) compared to wild-type control mice. Furthermore, the number of OCs was decreased (48.8-65.3% reduction) in Pax5Tg mice. These findings indicate that Pax5 plays a negative role in OC lineage specification and commitment through Blimp1 downregulation. Thus, our data suggest that the Pax5-Blimp1 axis is crucial for the regulation of RANKL-induced osteoclastogenesis.

T-Cell Death Associated Gene 51 Is a Novel Negative Regulator of PPARγ That Inhibits PPARγ-RXRα Heterodimer Formation in Adipogenesis

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator in adipogenesis. PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPARγ-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPARγ in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPARγ. The heterodimer formation of PPARγ-RXRα was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPARγ. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPARγ by blocking RXRα recruitment to the PPARγ-RXRα heterodimer complex in adipogenesis.

Engineered Recombinant PON1-OPH Fusion Hybrids: Potentially Effective Catalytic Bioscavengers against Organophosphorus Nerve Agent Analogs.

Organophosphorus nerve agents (OPNAs), including both G- and V-type nerve agents such as sarin, soman, tabun and VX, are extremely neurotoxic organophosphorus compounds. Catalytic bioscavengers capable of hydrolyzing OPNAs are under development because of the low protective effects and adverse side effects of chemical antidotes to OPNA poisoning. However, these bioscavengers have certain limitations for practical application, including low catalytic activity and narrow specificity. In this study, we generated a fusion-hybrid form of engineered recombinant human paraoxonase 1 (rePON1) and bacterial organophosphorus hydrolase (OPH), referred to as GV-hybrids, using a flexible linker to develop more promising catalytic bioscavengers against a broad range of OPNAs. These GV-hybrids were able to synergistically hydrolyze both G-type OPNA analogs (paraoxon: 1.7 ~ 193.7-fold, p-nitrophenyl diphenyl phosphate (PNPDPP): 2.3 ~ 33.0-fold and diisopropyl fluorophosphates (DFP): 1.4 ~ 22.8-fold) and V-type OPNA analogs (demeton-Smethyl (DSM): 1.9 ~ 34.6-fold and malathion: 1.1 ~ 4.2-fold above) better than their individual enzyme forms. Among the GV-hybrid clones, the GV7 clone showed remarkable improvements in the catalytic activity toward both G-type OPNA analogs (kcat/Km (106 M-1 min-1): 59.8 ± 0.06 (paraoxon), 5.2 ± 0.02 (PNPDPP) and 47.0 ± 6.0 (DFP)) and V-type OPNA analogs (kcat/Km (M-1 min-1): 504.3 ± 48.5 (DSM) and 1324.0 ± 47.5 (malathion)). In conclusion, we developed GV-hybrid forms of rePON1 and bacterial OPH mutants as effective and suitable catalytic bioscavengers to hydrolyze a broad range of OPNA analogs.

TDAG51 is a crucial regulator of maternal care and depressive-like behavior after parturition.

Postpartum depression is a severe emotional and mental disorder that involves maternal care defects and psychiatric illness. Postpartum depression is closely associated with a combination of physical changes and physiological stress during pregnancy or after parturition in stress-sensitive women. Although postpartum depression is relatively well known to have deleterious effects on the developing fetus, the influence of genetic risk factors on the development of postpartum depression remains unclear. In this study, we discovered a novel function of T cell death-associated gene 51 (TDAG51/PHLDA1) in the regulation of maternal and depressive-like behavior. After parturition, TDAG51-deficient dams showed impaired maternal behavior in pup retrieving, nursing and nest building tests. In contrast to the normal dams, the TDAG51-deficient dams also exhibited more sensitive depressive-like behaviors after parturition. Furthermore, changes in the expression levels of various maternal and depressive-like behavior-associated genes regulating neuroendocrine factor and monoamine neurotransmitter levels were observed in TDAG51-deficient postpartum brain tissues. These findings indicate that TDAG51 plays a protective role against maternal care defects and depressive-like behavior after parturition. Thus, TDAG51 is a maternal care-associated gene that functions as a crucial regulator of maternal and depressive-like behavior after parturition.

3,5-Di-C-ß-D-glucopyranosyl phloroacetophenone, a major component of Melicope ptelefolia, suppresses fibroblast activation and alleviates arthritis in a mouse model: Potential therapeutics for rheumatoid arthritis.

Melicope ptelefolia has been traditionally used to treat rheumatism and fever. The present study aimed to investigate the therapeutic effect of 3,5­di­C­ß­D­glucopyranosyl phloroacetophenone (ßGP), a main component of M. ptelefolia, on rheumatoid arthritis (RA). A model of collagen­induced arthritis (CIA) was established in mice using the RAW 264.7 murine macrophage cell line and mouse embryonic fibroblasts (MEFs). The clinical scores of arthritis, swelling, histopathological findings, and micro­computed tomography in CIA mouse paws were assessed. The levels of anti­type II collagen antibody and cytokines were determined in the plasma and cell culture supernatant, respectively. Protein and gene expression levels were analyzed by western blot and reverse transcription­quantitative polymerase chain reaction analyses. ßGP significantly decreased the gross arthritic scores of CIA mice and joint swelling, and decreased articular inflammation, cartilage degradation and bone erosion. However, ßGP did not exert any effect on anti­type II collagen immunoglobulin G plasma levels or inflammatory cytokine expression in macrophages. ßGP significantly suppressed the expression of interleukin­6 and leukemia inhibitory factor and decreased the phosphorylation of signal transducer and activator of transcription 3, and expression of receptor activator of nuclear factor­κB ligand in tumor necrosis factor­α­stimulated MEFs and in CIA mouse paws. Osteoclast­related gene expression was significantly reduced in CIA mouse paws. Taken together, ßGP suppressed the development of RA by regulating the activation of synovial fibroblasts.

Regulation of Osteoclast Differentiation by Cytokine Networks.

Cytokines play a pivotal role in maintaining bone homeostasis. Osteoclasts (OCs), the sole bone resorbing cells, are regulated by numerous cytokines. Macrophage colony-stimulating factor and receptor activator of NF-κB ligand play a central role in OC differentiation, which is also termed osteoclastogenesis. Osteoclastogenic cytokines, including tumor necrosis factor-α, IL-1, IL-6, IL-7, IL-8, IL-11, IL-15, IL-17, IL-23, and IL-34, promote OC differentiation, whereas anti-osteoclastogenic cytokines, including interferon (IFN)-α, IFN-ß, IFN-γ, IL-3, IL-4, IL-10, IL-12, IL-27, and IL-33, downregulate OC differentiation. Therefore, dynamic regulation of osteoclastogenic and anti-osteoclastogenic cytokines is important in maintaining the balance between bone-resorbing OCs and bone-forming osteoblasts (OBs), which eventually affects bone integrity. This review outlines the osteoclastogenic and anti-osteoclastogenic properties of cytokines with regard to osteoimmunology, and summarizes our current understanding of the roles these cytokines play in osteoclastogenesis.

Generation of an osteoblast-based artificial niche that supports in vitro B lymphopoiesis.

B lymphocytes are produced from hematopoietic stem cells (HSCs) through the highly ordered process of B lymphopoiesis, which is regulated by a complex network of cytokines, chemokines and cell adhesion molecules derived from the hematopoietic niche. Primary osteoblasts function as an osteoblastic niche (OBN) that supports in vitro B lymphopoiesis. However, there are significant limitations to the use of primary osteoblasts, including their relative scarcity and the consistency and efficiency of the limited purification and proliferation of these cells. Thus, development of a stable osteoblast cell line that can function as a biomimetic or artificial OBN is necessary. In this study, we developed a stable osteoblastic cell line, designated OBN4, which functions as an osteoblast-based artificial niche that supports in vitro B lymphopoiesis. We demonstrated that the production of a B220+ cell population from Lineage- (Lin-) Sca-1+ c-Kit+ hematopoietic stem and progenitor cells (HSPCs) was increased ~1.7-fold by OBN4 cells relative to production by primary osteoblasts and OP9 cells in coculture experiments. Consistently, OBN4 cells exhibited the highest production of B220+ IgM+ cell populations (6.7±0.6-13.6±0.6%) in an IL-7- and stromal cell-derived factor 1-dependent manner, with higher production than primary osteoblasts (3.7±0.5-6.4±0.6%) and OP9 cells (1.8±0.6-3.9±0.5%). In addition, the production of B220+ IgM+ IgD+ cell populations was significantly enhanced by OBN4 cells (15.4±1.1-18.9±3.2%) relative to production by primary osteoblasts (9.5±0.6-14.6±1.6%) and OP9 cells (9.1±0.5-10.3±1.8%). We conclude that OBN4 cells support in vitro B lymphopoiesis of Lin- Sca-1+ c-Kit+ HSPCs more efficiently than primary osteoblasts or OP9 stromal cells.

Improved Hydrolysis of Organophosphorus Compounds by Engineered Human Prolidases.

BACKGROUND: Human prolidase has weak hydrolytic activity for toxic organophosphorus compounds including diisopropyl fluorophosphates (DFP), chemical warfare nerve agents and pesticides. OBJECTIVES: In order to use human prolidase as a catalytic bioscavenger against toxic organophosphorus compound exposure, protein engineering is an important issue to improve the catalytic activity of human prolidase towards the hydrolysis of toxic organophosphorus compounds. METHOD: We developed two human prolidase mutants, A252R and P365R, with a single amino acid substitution using in silico analysis based on the sequence, protein structure and stability to improve the catalytic activity of human prolidase towards DFP hydrolysis. RESULTS: Our results showed that the catalytic efficiencies of A252R and P365R towards DFP hydrolysis were 1.23- and 1.36-fold increases, respectively, than that of the wild type, while the prolidase activities of A252R and P365R towards Leu-Pro hydrolysis were 0.88- and 0.78-fold decreases that of the wild type, respectively, indicating that substitution mutations of A252R and P365R in human prolidase show improved hydrolytic activity for toxic organophosphorus compounds. CONCLUSION: We report here that by introducing either the A252R or P365R substitution mutation, the structural changes affecting catalytic turnover rate and substrate binding affinity are valuable in improving the catalytic activity of human prolidase towards toxic organophosphorus compound hydrolysis.

Porcine amino peptidase N domain VII has critical role in binding and entry of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) infects swine intestinal cells causing enteric disease. Research has shown that the entry into these cells is through porcine aminopeptidase N (pAPN) receptor. To gain insights into mechanisms of PEDV-pAPN interactions, the present study aimed at identifying the domain that is critical for PEDV binding. To this end, NIH3T3 cell lines constitutively expressing pAPN or pAPN mutants were generated. The mutants were; domain VII deletion mutant and domains IV-VI deletion mutant. In the latter, domain VII was linked to the transmembrane segment through domain III. Results showed PEDV infection was restricted to pAPN and pAPN domain VII expressing NIH3T3 cells. Further, reducing PEDV titre 10 fold resulted in 37.8% decrease in foci indicating positive correlation. A time course test at 12, 24, 36, 48 and 60h showed that foci increased 6 fold in the overall time range. Also, PEDV harvested from pAPN or domain VII expressing NIH3T3 cells was induced indirect plaques in Vero cells confirming successful entry and replication. Collectively, our results demonstrate that PEDV recognizes pAPN and that the main interactive point is lodged within domain VII of the pAPN. These findings are important for therapeutic development as well as creating a platform for future studies on PEDV.