Monoclonal antibody Y01 prevents tauopathy progression induced by lysine 280-acetylated tau in cell and mouse models.

The spatiotemporal pattern of the spread of pathologically modified tau through brain regions in Alzheimer's disease (AD) can be explained by prion-like cell-to-cell seeding and propagation of misfolded tau aggregates. Hence, to develop targeted therapeutic antibodies, it is important to identify the seeding- and propagation-competent tau species. The hexapeptide 275VQIINK280 of tau is a critical region for tau aggregation, and K280 is acetylated in various tauopathies, including AD. However, the mechanism that links tau acetylated on lysine 280 (tau-acK280) to subsequent progression to neurodegenerative disease remains unclear. Here, we demonstrate that tau-acK280 is critical for tau propagation processes including secretion, aggregation, and seeding. We developed an antibody, Y01, that specifically targets tau-acK280 and solved the crystal structure of Y01 in complex with an acK280 peptide. The structure confirmed that Y01 directly recognizes acK280 and the surrounding residues. Strikingly, upon interaction with acetylated tau aggregates, Y01 prevented tauopathy progression and increased neuronal viability in neuron cultures and in tau-Tg mice through antibody-mediated neutralization and phagocytosis, respectively. Based on our observations that tau-acK280 is a core species involved in seeding and propagation activities, the Y01 antibody that specifically recognizes acK280 represents a promising therapeutic candidate for AD and other neurodegenerative diseases associated with tauopathy.

Enhanced cell growth, production, and mAb quality produced in Chinese hamster ovary-K1 cells by supplementing polyamine in the media.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing.

Antimicrobial peptides ß-defensin family: Expression and regulation in the endometrium during the estrous cycle and pregnancy in pigs.

Members of the ß-defensin (DEFB) family, which are antimicrobial peptides and humoral components of the innate immune system, protect the surfaces of various host tissues by killing a broad range of microorganisms and are involved in immunomodulatory actions. The expression of these DEFB members changed during the estrous cycle and pregnancy in a stage-specific manner. The expression of DEFBs was also detected in conceptus and chorioallantoic tissues during pregnancy. DEFB1 and DEFB3 proteins and DEFB2 mRNA were localized primarily to endometrial epithelial cells during early pregnancy. Increasing doses of progesterone upregulated DEFB2 and EP2C expression in endometrial explant tissues. These results showed that members of the DEFB family were expressed stage-specifically at the maternal-conceptus interface in pigs, suggesting that the DEFB family plays important roles at the maternal-conceptus interface in regulation of innate immunity by protection of the maternal endometrial and conceptus tissues from pathogens to preserve fertility in pigs.

Effect of teriparatide on drug treatment of tuberculous spondylitis: an experimental study.

Tuberculous spondylitis often develops catastrophic bone destruction with uncontrolled inflammation. Because anti-tuberculous drugs do not have a role in bone formation, a combination drug therapy with a bone anabolic agent could help in fracture prevention and promote bone reconstruction. This study aimed to investigate the influence of teriparatide on the effect of anti-tuberculous drugs in tuberculous spondylitis treatment. We used the virulent Mycobacterium tuberculosis (Mtb) H37Rv strain. First, we investigated the interaction between teriparatide and anti-tuberculosis drugs (isoniazid and rifampin) by measuring the minimal inhibitory concentration (MIC) against H37Rv. Second, we evaluated the therapeutic effect of anti-tuberculosis drugs and teriparatide on our previously developed in vitro tuberculous spondylitis model of an Mtb-infected MG-63 osteoblastic cell line using acid-fast bacilli staining and colony-forming unit counts. Selected chemokines (interleukin [IL]-8, interferon γ-induced protein 10 kDa [IP-10], monocyte chemoattractant protein [MCP]-1, and regulated upon activation, normal T cell expressed and presumably secreted [RANTES]) and osteoblast proliferation (alkaline phosphatase [ALP] and alizarin red S [ARS] staining) were measured. Teriparatide did not affect the MIC of isoniazid and rifampin. In the Mtb-infected MG-63 spondylitis model, isoniazid and rifampin treatment significantly reduced Mtb growth, and cotreatment with teriparatide did not change the anti-tuberculosis effect of isoniazid (INH) and rifampin (RFP). IP-10 and RANTES levels were significantly increased by Mtb infection, whereas teriparatide did not affect all chemokine levels as inflammatory markers. ALP and ARS staining indicated that teriparatide promoted osteoblastic function even with Mtb infection. Cotreatment with teriparatide and the anti-tuberculosis drugs activated bone formation (ALP-positive area increased by 705%, P = 0.0031). Teriparatide was effective against Mtb-infected MG63 cells without the anti-tuberculosis drugs (ARS-positive area increased by 326%, P = 0.0037). Teriparatide had no effect on the efficacy of anti-tuberculosis drugs and no adverse effect on the activity of Mtb infection in osteoblasts. Furthermore, regulation of representative osteoblastic inflammatory chemokines was not changed by teriparatide treatment. In the in vitro Mtb-infected MG-63 cell model of tuberculous spondylitis, cotreatment with the anti-tuberculosis drugs and teriparatide increased osteoblastic function.

Structure-based molecular characterization of the YetL transcription factor from Bacillus subtilis.

Bacillus subtilis is a gram-positive bacterium that has developed to coordinate gene expression and to survive against changes of nutrients and toxic chemicals. Flavonoids are exuded by plant cells and are abundant in the soil. To counteract the antibacterial effects of flavonoids, B. subtilis expresses flavonoid-detoxifying enzymes, and their expression is negatively regulated by transcription factors, including YetL. YetL was shown to control B. subtilis growth through the promoter regions of yetL and yetM genes in response to some flavonoids. Despite the functional significance of the YetL transcription factor in bacterial survival, no structural information is available for YetL. Here, we report the crystal structure of YetL and propose a flavonoid-induced regulatory mechanism. The YetL structure contains the canonical winged helix-turn-helix motif of the MarR superfamily but distinctly presents an additional N-terminal helix. In the dimeric assembly of YetL, the H1 helix intersects the YetL dimer and contributes to extensive intersubunit interactions. As a transcription factor, YetL recognizes a 28-mer operator of double-stranded DNA that contains a palindromic sequence. Moreover, our comparative structural analysis of YetL and other MarR members allows us to propose a flavonoid-induced transcription regulatory mechanism that is used for bacterial adaptation to environmental changes and stresses.

Structure-based molecular characterization of the LltR transcription factor from Listeria monocytogenes.

Listeria monocytogenes is a psychrotrophic food-borne pathogenic bacterium that causes listeriosis. Due to its unusual adaptation, an ability to grow at extended temperatures ranging from 4 to 45 °C, L. monocytogenes is notoriously hard to control in food-manufacturing processes. In addition, the growing number of antibiotic-resistant L. monocytogenes strains have made listeriosis steadily refractory to clinical treatments and can lead to serious life-threatening diseases, such as sepsis and meningitis, in immunocompromised persons and neonates. Transcription factors that belong to the PadR family play a key role in bacterial survival against unfavorable environmental stresses. The LltR protein from L. monocytogenes was identified as a PadR-type transcription factor and was shown to be required for bacterial growth adaptation at low temperatures. Despite the functional significance of LltR in listeria survival and pathogenesis, our molecular understanding of the LltR-mediated transcriptional regulation is highly limited. Here, we report the crystal structure of LltR and reveal the operator DNA recognition mechanism used by LltR. LltR dimerizes into an isosceles triangle-like shape and requires a winged helix-turn-helix motif for dsDNA recognition. Indeed, LltR and putative operator dsDNA binding was observed and suggests a transcriptional repression of the llfR-lmo0600-lmo0601 operon by direct interaction between the LltR transcription factor and its promoter region. Structure-based comparative and mutational analyses showed that LltR interacts with dsDNA via a unique strategy that combines both LltR-specific and PadR family-common mechanisms.

A Rapid Assessing Method of Drug Susceptibility Using Flow Cytometry for Mycobacterium tuberculosis Isolates Resistant to Isoniazid, Rifampin, and Ethambutol.

BACKGROUND: The current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed. METHODS: To evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results. RESULTS: The susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST. CONCLUSION: In conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.

Fungal ß-Glycosidase Belonging to Subfamily 4 of Glycoside Hydrolase Family 30 with Transglycosylation Activity.

Fomitopsis palustris, a prominent wood decayer, is known to produce a variety of glycoside hydrolases (GHs). In this study, we characterized a fungal ß-glycosidase belonging to subfamily 4 of GH family 30 (GH30). The recombinant protein (FpGH30) showed the highest hydrolytic activity toward p-nitrophenyl-ß-d-fucopyranoside (pNPßFuc), followed by p-nitrophenyl-α-l-arabinopyranoside (pNPαAra) and p-nitrophenyl-ß-d-galactopyranoside (pNPßGal). FpGH30 also exhibited transglycosylation activities, which catalyzed the transfer of glycosyl moieties to different glycosides and alkyl alcohols. When pNPßFuc, pNPßGal, and pNPαAra were used as substrates, self-condensation reactions occurred, leading to the production of the corresponding transglycosylated products with yields of 21, 26, and 25%, respectively. The enzyme was also able to catalyze the transfucosylation of pNP derivatives of ß-d-glucose, ß-d-mannose, and ß-d-xylose and alkyl alcohols (C1-C6), producing the corresponding transfucosylated products and alkyl fucosides. Our study indicates that FpGH30 is the first characterized fungal ß-glycosidase belonging to subfamily 4 of GH30 with transglycosylation activities.

Expression of Caspases in the Pig Endometrium Throughout the Estrous Cycle and at the Maternal-Conceptus Interface During Pregnancy and Regulation by Steroid Hormones and Cytokines.

Caspases, a family of cysteine protease enzymes, are a critical component of apoptotic cell death, but they are also involved in cellular differentiation. The expression of caspases during apoptotic processes in reproductive tissues has been shown in some species; however, the expression and regulation of caspases in the endometrium and placental tissues of pigs has not been fully understood. Therefore, we determined the expression of caspases CASP3, CASP6, CASP7, CASP8, CASP9, and CASP10 in the endometrium throughout the estrous cycle and pregnancy. During the estrous cycle, the expression of all caspases and during pregnancy, the expression of CASP3, CASP6, and CASP7 in the endometrium changed in a stage-specific manner. Conceptus and chorioallantoic tissues also expressed caspases during pregnancy. CASP3, cleaved-CASP3, and CASP7 proteins were localized to endometrial cells, with increased levels in luminal and glandular epithelial cells during early pregnancy, whereas apoptotic cells in the endometrium were limited to some scattered stromal cells with increased numbers on Day 15 of pregnancy. In endometrial explant cultures, the expression of some caspases was affected by steroid hormones (estradiol-17ß and/or progesterone), and the cytokines interleukin-1ß and interferon-γ induced the expression of CASP3 and CASP7, respectively. These results indicate that caspases are dynamically expressed in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy in response to steroid hormones and conceptus signals. Thus, caspase action could be important in regulating endometrial and placental function and epithelial cell function during the implantation period in pigs.

Structure-based functional analysis of a PadR transcription factor from Streptococcus pneumoniae and characteristic features in the PadR subfamily-2.

Since the first discovery of phenolic acid decarboxylase transcriptional regulator (PadR), its homologs have been identified mostly in bacterial species and constitute the PadR family. PadR family members commonly contain a winged helix-turn-helix (wHTH) motif and function as a transcription factor. However, the PadR family members are varied in terms of molecular size and structure. As a result, they are divided into PadR subfamily-1 and PadR subfamily-2. PadR subfamily-2 proteins have been reported in some pathogenic bacteria, including Listeria monocytogenes and Streptococcus pneumoniae, and implicated in drug resistance processes. Despite the growing numbers of known PadR family proteins and their critical functions in bacteria survival, biochemical and biophysical studies of the PadR subfamily-2 are limited. Here, we report the crystal structure of a PadR subfamily-2 member from Streptococcus pneumoniae (SpPadR) at a 2.40 Å resolution. SpPadR forms a dimer using its N-terminal and C-terminal helices. The two wHTH motifs of a SpPadR dimer expose their positively charged residues presumably to interact with DNA. Our structure-based mutational and biochemical study indicates that SpPadR specifically recognizes a palindromic nucleotide sequence upstream of its encoding region as a transcriptional regulator. Furthermore, comparative structural analysis of diverse PadR family members combined with a modeling study highlights the structural and regulatory features of SpPadR that are canonical to the PadR family or specific to the PadR subfamily-2.

Toll-like Receptor 5 Activation by the CagY Repeat Domains of Helicobacter pylori.

Helicobacter pylori (Hp) is an important human pathogen associated with gastric inflammation and neoplasia. It is commonly believed that this bacterium avoids major immune recognition by Toll-like receptors (TLRs) because of low intrinsic activity of its flagellin and lipopolysaccharides (LPS). In particular, TLR5 specifically detects flagellins in various bacterial pathogens, while Hp evolved mutations in flagellin to evade detection through TLR5. Cancerogenic Hp strains encode a type IV secretion system (T4SS). The T4SS core component and pilus-associated protein CagY, a large VirB10 ortholog, drives effector molecule translocation. Here, we identify CagY as a flagellin-independent TLR5 agonist. We detect five TLR5 interaction sites, promoting binding of CagY-positive Hp to TLR5-expressing cells, TLR5 stimulation, and intracellular signal transduction. Consequently, CagY constitutes a remarkable VirB10 member detected by TLR5, driving crucial innate immune responses by this human pathogen.

Transcriptomic analysis of interferon-γ-regulated genes in endometrial explants and their possible role in regulating maternal endometrial immunity during the implantation period in pigs, a true epitheliochorial placentation species.

The implantation process requires precisely controlled interactions between the maternal uterine endometrium and the implanting conceptus. Conceptus-derived secretions affect endometrial cells to facilitate the adhesion and attachment of trophoblasts, and endometrial secretions support the growth and development of the conceptus. In pigs, the conceptus secretes a large amount of type II interferon, interferon-γ (IFNG), during the implantation period. However, the role of IFNG in the implantation process has not been fully understood in pigs. Thus, to determine the role of IFNG in the endometrium during early pregnancy in pigs, we treated endometrial explant tissues with increasing doses of IFNG and analyzed the transcriptome regulated by IFNG using an RNA-sequencing analysis. Data analyses identified 276 differentially regulated genes, their Gene Ontology terms, and 94 signature genes in a Gene Set Enrichment Analysis. Furthermore, we analyzed the expression of IFNG-regulated genes, including CIITA, KYNU, IDO1, WARS, and MHC class II molecules, in the endometrium throughout pregnancy and found that levels of those genes in the endometrium were highest on Day 15 of pregnancy, corresponding to the time of peak IFNG secretion by porcine conceptuses. In addition, immunohistochemical analyses revealed that CIITA, KYNU, and IDO proteins were expressed in a cell type- and pregnancy status-specific manner in the endometrium. These results show that genes overrepresented in endometrial tissues in response to IFNG were mainly related to immune responses, suggesting that conceptus-derived IFNG could play critical roles in regulating the maternal immune response for the establishment of pregnancy in pigs.

Crystal structure of the Pseudomonas aeruginosa PA0423 protein and its functional implication in antibiotic sequestration.

Pseudomonas aeruginosa is a widely found opportunistic pathogen. The emergence of multidrug-resistant strains and persistent chronic infections have increased. The protein encoded by the pa0423 gene in P. aeruginosa is proposed to be critical for pathogenesis and could be a virulence-promoting protease or a bacterial lipocalin that binds a lipid-like antibiotic for drug resistance. Although two functions of proteolysis and antibiotic resistance are mutually related to bacterial survival in the host, it is very unusual for a single-domain protein to target unrelated ligand molecules such as protein substrates and lipid-like antibiotics. To clearly address the biological role of the PA0423 protein, we performed structural and biochemical studies. We found that PA0423 adopts a single-domain ß-barrel structure and belongs to the lipocalin family. The PA0423 structure houses an internal tubular cavity, which accommodates a ubiquinone-8 molecule. Furthermore, we reveal that PA0423 can directly interact with the polymyxin B antibiotic using the internal cavity, suggesting that PA0423 has a physiological function in the antibiotic resistance of P. aeruginosa.

Uterine epithelial expression of the tumor necrosis factor superfamily: a strategy for immune privilege during pregnancy in a true epitheliochorial placentation species.

The maternal immune system tolerates semi-allogeneic placental tissues during pregnancy. Fas ligand (FASLG) and tumor necrosis factor superfamily 10 (TNFSF10) are known to be components of maternal immune tolerance in humans and mice. However, the role of FASLG and TNFSF10 in the tolerance process has not been studied in pigs, which form a true epitheliochorial type placenta. Thus, the present study examined the expression and function of FASLG and TNFSF10 and their receptors at the maternal-conceptus interface in pigs. The endometrium and conceptus tissues expressed FASLG and TNFSF10 and their receptor mRNAs during pregnancy in a stage-specific manner. During pregnancy, FASLG and TNFSF10 proteins were localized predominantly to endometrial luminal epithelial cells with strong signals on Day 30 to term and on Day 15, respectively, and receptors for TNFSF10 were localized to some stromal cells. Interferon-γ (IFNG) increased the expression of TNFSF10 and FAS in endometrial tissues. Co-culture of porcine endometrial epithelial cells over-expressing TNFSF10 with peripheral blood mononuclear cells yielded increased apoptotic cell death of lymphocytes and myeloid cells. In addition, many apoptotic T cells were found in the endometrium on Day 15 of pregnancy. The present study demonstrated that FASLG and TNFSF10 were expressed at the maternal-conceptus interface and conceptus-derived IFNG increased endometrial epithelial TNFSF10, which, in turn, induced apoptotic cell death of immune cells. These results suggest that endometrial epithelial FASLG and TNFSF10 may be critical for the formation of micro-environmental immune privilege at the maternal-conceptus interface for the establishment and maintenance of pregnancy in pigs.

T4SS-dependent TLR5 activation by Helicobacter pylori infection.

Toll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Highly virulent H. pylori strains possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5+ epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5-knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori.

Structure-based molecular characterization and regulatory mechanism of the LftR transcription factor from Listeria monocytogenes: Conformational flexibilities and a ligand-induced regulatory mechanism.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis and can lead to serious clinical problems, such as sepsis and meningitis, in immunocompromised patients and neonates. Due to a growing number of antibiotic-resistant L. monocytogenes strains, listeriosis can steadily become refractory to antibiotic treatment. To develop novel therapeutics against listeriosis, the drug resistance mechanism of L. monocytogenes needs to be determined. The transcription factor LftR from L. monocytogenes regulates the expression of a putative multidrug resistance transporter, LieAB, and belongs to the PadR-2 subfamily of the PadR family. Despite the functional significance of LftR, our molecular understanding of the transcriptional regulatory mechanism for LftR and even for the PadR-2 subfamily is highly limited. Here, we report the crystal structure of LftR, which forms a dimer and protrudes two winged helix-turn-helix motifs for DNA recognition. Structure-based mutational and comparative analyses showed that LftR interacts with operator DNA through a LftR-specific mode as well as a common mechanism used by the PadR family. Moreover, the LftR dimer harbors one intersubunit cavity in the center of the dimeric structure as a putative ligand-binding site. Finally, conformational flexibilities in the LftR dimer and in the cavity suggest that a ligand-induced regulatory mechanism would be used by the LftR transcription factor.

Analysis of stage-specific expression of the toll-like receptor family in the porcine endometrium throughout the estrous cycle and pregnancy.

Toll-like receptors (TLRs) play critical roles in innate immunity by regulating antimicrobial responses in mucosal tissues. The expression and function of TLRs in female reproductive tissues have been studied in several species, but the expression and function of TLRs and MYD88, an adaptor molecule in the TLR signaling pathway, at the maternal-conceptus interface are not well understood in pigs. Thus, we determined the expression of TLR1 - TLR10 and MYD88 in the endometrium, conceptus, and chorioallantoic tissues of pigs. TLR1 - TLR10 and MYD88 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy in a stage-dependent manner. TLR and MYD88 mRNAs were also detected in early stage conceptuses and chorioallantoic tissues from Day 30 to term pregnancy. The expression of TLR2, TLR4, TLR5, and TLR7 was localized to epithelial and stromal cells in endometrial and chorioallantoic tissues. Increasing doses of P4, but not E2, induced the expression of TLR4, TLR5, TLR6, TLR7, and TLR8, while interferon-γ increased the expression of TLR2 and TLR7 in endometrial explant tissues. Expression of TLR3, TLR5, TLR6, TLR7, and MYD88 was higher in the endometrium with somatic cell nucleus transfer-derived conceptuses than conceptuses derived from natural mating on Day 12. These results indicate that the expression of TLR1 - TLR10 and MYD88 is dynamically regulated at the maternal-conceptus interface in pigs, suggesting that TLRs expressed in the endometrium and the placenta may play a critical role in regulating mucosal immune responses to support the establishment and maintenance of pregnancy.

Crystal structure of Bacillus cereus flagellin and structure-guided fusion-protein designs.

Flagellin is a major component of the flagellar filament. Flagellin also functions as a specific ligand that stimulates innate immunity through direct interaction with Toll-like receptor 5 (TLR5) in the host. Because flagellin activates the immune response, it has been of interest to develop as a vaccine adjuvant in subunit vaccines or antigen fusion vaccines. Despite the widespread application of flagellin fusion in preventing infectious diseases, flagellin-antigen fusion designs have never been biophysically and structurally characterized. Moreover, flagellin from Salmonella species has been used extensively despite containing hypervariable regions not required for TLR5 that can cause an unexpected immune response. In this study, flagellin from Bacillus cereus (BcFlg) was identified as the smallest flagellin molecule containing only the conserved TLR5-activating D0 and D1 domains. The crystal structure of BcFlg was determined to provide a scheme for fusion designs. Through homology-based modeling and comparative structural analyses, diverse fusion strategies were proposed. Moreover, cellular and biophysical analysis of an array of fusion constructs indicated that insertion fusion at BcFlg residues 178-180 does not interfere with the protein stability or TLR5-stimulating capacity of flagellin, suggesting its usefulness in the development and optimization of flagellin fusion vaccines.

Expression and regulation of inhibitor of DNA binding proteins ID1, ID2, ID3, and ID4 at the maternal-conceptus interface in pigs.

Inhibitor of DNA binding (ID) proteins, ID1, ID2, ID3, and ID4 are transcriptional regulators that have a helix-loop-helix (HLH) domain but not a basic DNA binding domain. ID proteins inhibit the functions of basic HLH transcription factors and regulate cell proliferation and differentiation. However, the expression and function of ID1, ID2, ID3, and ID4 at the maternal-conceptus interface are not fully understood in pigs. Therefore, we determined the expressions of ID1, ID2, ID3, and ID4 in porcine endometrium, conceptus, and chorioallantoic tissues. ID1, ID2, ID3, and ID4 mRNAs were expressed in the endometrium, with lower levels of ID1, ID2, and ID4 on Day 12 of pregnancy than during the estrous cycle. ID1, ID2, ID3, and ID4 mRNAs were also detected in conceptus and chorioallantoic tissues during pregnancy. ID2 protein was mainly localized to luminal epithelia and weakly to vascular smooth muscle cells in the endometrium and conceptus trophectoderm. Increasing doses of interleukin-1ß decreased levels of ID2 mRNA, while estradiol-17ß increased levels of ID3 mRNA in endometrial explants. The expressions of ID2 and ID4 mRNAs were higher in endometria from gilts with somatic cell nuclear transfer-derived conceptuses compared with endometria from gilts carrying conceptuses derived from natural mating on Day 12. These results indicate that the expressions of ID family genes are dynamically regulated at the maternal-conceptus interface, suggesting that ID proteins may play critical roles in the regulation of endometrial epithelial cell function and conceptus development to support the establishment and maintenance of pregnancy in pigs.