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The aim of the present study was to investigate the effect of glycosylation with sugar beet pectin (SBP) on the interfacial behaviour and emulsifying ability of coconut protein (CP). The physical stabilities of the emulsions were predicted by transmission variation, droplet distribution and zeta potentials. The results showed that SBP-CP-stabilized emulsions showed better stability during centrifugation than those stabilized by CP because SBP-CP reduced the degree of variation in the CP transmission profile. The adsorption kinetics of all emulsifiers at the oil-water interface were determined to investigate the relationship between the interfacial behaviour and emulsion stability. The presence of SBP considerably reduced the adsorption rate of CP (0.698 mN/m/s1/2) and hampered the development of a highly viscoelastic network at the oil-water interface. The values of the dilatational elastic modulus (Ed = 19.477 mN/m) and dilatational viscous modulus (E = 19.719 mN/m) were approximately equal, indicating that the adsorption process was mainly dominated by elastic behaviour. Additionally, the SBP-CP interaction enhanced the dilatational property of the CP-absorbed layer.

Immunomolecular characterization of Ehrlichia chaffeensis (E. ch.) and E. canis (E. ca.) has defined protein orthologs, including tandem repeat proteins (TRPs) that have immunodominant linear antibody epitopes. In this study, we combined bioinformatic analysis and cell-free protein expression to identify undiscovered immunoreactive E. ch. and E. ca. hypothetical proteins. Antigenicity of the E. ch. and E. ca. ORFeomes (n = 1105 and n = 925, respectively) was analyzed by the sequence-based prediction model ANTIGENpro, and we identified ~250 ORFs in each respective ORFeome as highly antigenic. The hypothetical proteins (E. ch. n = 93 and E. ca. n = 98) present in the top 250 antigenic ORFs were further investigated in this study. By ELISA, 46 E. ch. and 30 E. ca. IVTT-expressed hypothetical proteins reacted with antibodies in sera from naturally E. ch.-infected patients or E. ca.-infected dogs. Moreover, 15 E. ch. and 16 E. ca. proteins consistently reacted with a panel of sera from patients or dogs, including many that revealed the immunoreactivity of “gold standard” TRPs. Antibody epitopes in most (>70%) of these proteins exhibited partial or complete conformation-dependence. The majority (23/31; 74%) of the major immunoreactive proteins identified were small (≤250 aa), and 20/31 (65%) were predicted to be secreted effectors. Unlike the strong linear antibody epitopes previously identified in TRP and OMP orthologs, there were contrasting differences in the E. ch. and E. ca. antigenic repertoires, epitopes and ortholog immunoreactivity. This study reveals numerous previously undefined immunodominant and subdominant antigens, and illustrates the breadth, complexity, and diversity of immunoreactive proteins/epitopes in Ehrlichia.

The immunomes of Ehrlichia chaffeensis and Ehrlichia canis have recently been revised to include immunodominant hypothetical proteins with conformational antibody epitopes. In this study, we examined 216 E. chaffeensis and 190 E. canis highly antigenic proteins according to ANTIGENpro and also performed a genome-wide hypothetical protein analysis (E. chaffeensis n = 104; E. canis n = 124) for immunoreactivity. Using cell-free protein expression and immunoanalysis, 118 E. chaffeensis and 39 E. canis proteins reacted with sera from naturally E. chaffeensis-infected patients or E. canis-infected dogs. Moreover, 22 E. chaffeensis and 18 E. canis proteins consistently and strongly reacted with a panel of patient or canine sera. A subset of E. chaffeensis (n = 18) and E. canis (n = 9) proteins were identified as immunodominant. Consistent with our previous study, most proteins were classified as hypothetical, and the antibody epitopes exhibited complete or partial conformation dependence. The majority (28/40, 70%) of E. chaffeensis and E. canis proteins contained transmembrane domains, and 19 (48%) were predicted to be secreted effectors. The antigenic repertoires of E. chaffeensis and E. canis were mostly diverse and suggest that the immunomes of these closely related ehrlichiae are dominated by species-specific conformational antibody epitopes. This study reveals a significant group of previously undefined E. chaffeensis and E. canis antigens and reaffirms the importance of conformation-dependent epitopes as targets of anti-Ehrlichia immune responses. These findings substantially expand our understanding of host-Ehrlichia immune responses, advance efforts to define the molecular features of protective proteins, and improve prospects for effective vaccines for the ehrlichioses.

The contribution of rheological properties and viscoelasticity of the interfacial adsorbed layer to the emulsification mechanism of enzymatic modified sugar beet pectin (SBP) was studied. The component content of each enzymatic modified pectin was lower than that of untreated SBP. Protein and ferulic acid decreased from 5.52% and 1.08% to 0.54% and 0.13%, respectively, resulting in a decrease in thermal stability, apparent viscosity, and molecular weight (Mw). The dynamic interfacial rheological properties showed that the interfacial pressure and modulus (E) decreased significantly with the decrease of functional groups (especially proteins), which also led to the bimodal distribution of particle size. These results indicated that the superior emulsification property of SBP is mainly determined by proteins, followed by ferulic acid, and the existence of other functional groups also promotes the emulsification property of SBP.

Ehrlichia chaffeensis expresses the TRP120 multifunctional effector, which is known to play a role in phagocytic entry, on the surface of infectious dense-cored ehrlichiae, but a cognate host receptor has not been identified. We recently reported that E. chaffeensis activates canonical Wnt signaling in monocytes to promote bacterial uptake and intracellular survival and that TRP120 was involved in this activation event. To identify the specific mechanism of pathway activation, we hypothesized that TRP120 is a Wnt signaling ligand mimetic that initiates Wnt pathway activity through direct interaction with the Wnt pathway Frizzled family of receptors. In this study, we used confocal immunofluorescence microscopy to demonstrate very strong colocalization between E. chaffeensis and Fzd2, 4, 5, 7, and 9 as well as coreceptor LRP5 at 1 to 3 h postinfection. Direct binding between TRP120 and multiple Fzd receptors was further confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Interfering RNA knockdown of Wnt receptors, coreceptors, and signaling pathway components significantly reduced E. chaffeensis infection, demonstrating that complex and redundant interactions are involved in Wnt pathway exploitation. We utilized in silico approaches to identify a repetitive short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands and used mutant SLiM peptides and an α-TRP120-Wnt-SLiM antibody to demonstrate that the TRP120 Wnt SLiM activates the canonical Wnt pathway and promotes E. chaffeensis infection. This study reports the first example of bacterial mimicry of Wnt pathway ligands and highlights a pathogenic mechanism with potential for targeting by antimicrobial therapeutics. IMPORTANCE Upon infecting mammalian hosts, Ehrlichia chaffeensis establishes a replicative niche in microbe-eating immune system cells where it expertly orchestrates infection and spread. One of the ways Ehrlichia survives within these phagocytes is by activating evolutionarily conserved signaling pathways including the Wnt pathway; however, the molecular details of pathway hijacking have not been defined. This study is significant because it identifies an ehrlichial protein that directly interacts with components of the Wnt receptor complex, influencing pathway activity and promoting infection. Consequentially, Ehrlichia serves as a unique tool to investigate the intricacies of how pathogens repurpose human immune cell signaling and provides an opportunity to better understand many cellular processes in health and disease. Furthermore, understanding how this bacterium utilizes its small genome to survive within cells that evolved to destroy pathogens will facilitate the development of antibacterial therapeutics that could target Ehrlichia as well as other intracellular agents of human disease.

Continuous cropping of the same crop leads to land degradation. This is also called the continuous‐cropping obstacle. Here, we investigated how long‐term continuous cropping of tobacco influences soil biochemical properties and bacterial networks in the mountain lands of China. Two different fields were sampled: one with 25 years of continuous cropping tobacco and one with noncontinuous cropping tobacco. Soil chemical and biological properties were measured including available phosphorus and potassium, soil organic matter, pH, alkali‐hydrolysable nitrogen, micronutrients contents, and activity of urease, catalase, invertase, and phosphatase as well as tobacco agronomic characteristics. Bacterial communities of the two different soils were sequenced by metabarcoding of the 16S ribosomal RNA, and, with these data, network analysis was done. Soil chemical properties and tobacco agronomical properties were negatively affected by the continuous‐cropping obstacle, and this treatment has a less complex network (less modules, nodes, and connectivity) than the soil with noncontinuous cropping treatment. For continuous cropping, there were less generalists, which were key species that connect network, than noncontinuous cropping. Moreover, the taxonomic composition of bacterial network was different in the two different treatments. In the continuous‐cropping network, 40% nodes had negative interactions, suggesting that more competition or antagonism existed among bacterial species. It concluded that continuous cropping has a detrimental effect on soil chemical and that the bacterial network properties under continuous cropping are more sensitive to soil variables (so more unstable and inefficient) because there are less bacterial species that interact each other and this is due to limited nutrients or excessive toxic nutrient.

International audience; Ehrlichia spp. are important tick-borne pathogens of animals in Brazil, and Ehrlichia canis is the most prevalent species infecting dogs. Moreover, Ehrlichia minasensis has also recently been identified as a novel ehrlichial agent that infects cattle in Brazil. The objective of this study was to determine whether dogs could be infected by E. minasensis. To investigate this possibility, sera (n = 429) collected from dogs in the Pantanal region were retrospectively analyzed for the presence of antibodies against E. canis and E. minasensis. Canine sera were screened by two isolates of E. canis in indirect immunofluorescence assay (IFA) and the majority (n = 298; 69.4%) had antibodies with endpoint titers ranging from 80 to 327,680. In order to further confirm E. canis-specific antibodies, IFA positive sera were analyzed by ELISA using E. canis-specific peptides (i.e. TRP19 and TRP36 US/BR/CR), which detected E. canis antibodies in 80.2% (239/298) of the dog sera. Fifty-nine (13.7%) samples had detectable antibodies to E. canis by IFA but were negative by E. canis peptide ELISA. These sera were then tested by E. minasensis IFA (Cuiaba strain) as antigen and 67.8% (40/59) were positive (titers ranging from 80 to 20,480). Eleven sera had antibody titers against E. minasensis at least two-fold higher than observed for E. canis and suggests that these dogs were previously infected with E. minasensis. The results of the present study suggest that multiple ehrlichial agents infect dogs in Brazil, which highlights the need to consider different Ehrlichia spp. in Brazilian dogs, particularly in areas where dogs are frequently exposed to multiple tick species. This investigation is the first to provide serologic evidence of E. minasensis infection in dogs from Brazil.

β-defensin (BD) is a cysteine-rich cationic antibacterial peptide that is active against a wide range of bacteria. Here, a β-defensin homolog (LcBD2) was identified in large yellow croaker (Larimichthys crocea). The open reading frame of LcBD2 contains 195 nucleotides, encoding a protein of 64 amino acids that possesses a typical arrangement of six conserved cysteine residues (C31 , C37 , C41 , C53 , C59 and C60 ). LcBD2 transcripts were constitutively expressed in all examined tissues and significantly increased in head kidney, spleen and gills by Vibrio alginolyticus. The synthetic LcBD2 peptide imparted antimicrobial effects on both Gram-negative bacteria (V. campbellii, V. parahaemolyticus, V. alginolyticus, V. harveyi and Pseudomonas plecoglossicida) and Gram-positive bacteria (Bacillus subtilis). We also observed that after treatment with synthetic LcBD2 peptide, numerous blisters appeared on the membrane of P. plecoglossicida, which in turn may result in cell membrane breakage and bacterial death. Moreover, the synthetic LcBD2 peptide significantly upregulated the expression levels of TNF-α2, IL-1β and CXCL8_L1 in monocytes/macrophages, while downregulated expression level of IL-10. The LcBD2 peptide also remarkedly enhanced the phagocytosis of monocytes/macrophages. These results indicate that LcBD2 not only protects large yellow croaker against multiple bacterial pathogens but also plays a role in activation of monocytes/macrophages.

Interferon regulatory factors (IRFs) are crucial transcription factors involved in transcriptional regulation of type I interferons (IFNs) and IFN-stimulated genes (ISGs) against viral infection. In teleost fish, eleven IRFs have been found, however, understanding of their roles in the antiviral response remains limited. In the previous study, IRF1 (LcIRF1) and IRF2 (LcIRF2) genes were cloned from large yellow croaker (Larimichthys crocea). Here, we further characterized their function in the antiviral response. LcIRF1 and LcIRF2 were constitutively expressed in primary head kidney monocytes/macrophages (PKMs), lymphocytes (PKLs), granulocytes (PKGs) and large yellow croaker head kidney (LYCK) cell line, and significantly upregulated in PKMs and LYCK cells after stimulation with poly (I:C). LcIRF1 could induce promoter activities of three large yellow croaker type I IFNs, IFNc, IFNd and IFNh, while LcIRF2 could only induce those of IFNd and IFNh, and inhibit IFNc promoter activity. Correspondingly, overexpression of LcIRF1 in LYCK cells increased expression of all three IFNs (IFNc, IFNd and IFNh), while that of LcIRF2 only upregulated the expression levels of IFNd and IFNh, and inhibited expression of IFNc, although both LcIRF1and LcIRF2 induced expression of IFN-stimulated genes (ISGs), MxA, PKR and Viperin. Additionally, both LcIRF1 and LcIRF2 inhibited the Spring Viremia of Carp Virus (SVCV) replication in epithelioma papulosum cyprinid (EPC) cells, thus showing antiviral activity. Taken together, these results indicated that both LcIRF1 and LcIRF2 play positive roles in regulating the antiviral response of large yellow croaker by induction of distinct subgroups of type I IFNs.

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulating interferon (IFN) responses and immune cell development. A total of 11 IRFs have been identified in teleost fish. Here, a complete repertoire of 11 IRFs (LcIRFs) in the large yellow croaker (Larimichthys crocea) was characterized with the addition of five newly identified members, LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11. These five LcIRFs possess a DNA-binding domain (DBD) at the N-terminal that contains five to six conserved tryptophan residues and an IRF-association domain (IAD) or IAD2 at the C-terminal that is responsible for interaction with other IRFs or co-modulators. Phylogenetic analysis showed that the 11 LcIRFs were divided into four clades including the IRF1 subfamily, IRF3 subfamily, IRF4 subfamily, and IRF5 subfamily. These are evolutionarily related to their respective counterparts in other fish species. The 11 LcIRFs were constitutively expressed in all examined tissues, although at different expression levels. Upon polyinosinic: polycytidylic acid (poly (I:C)) stimulation, the expression of all 11 LcIRFs was significantly induced in the head kidney and reached the highest levels at 6 h post-stimulation (except LcIRF4). LcIRF1, LcIRF3, LcIRF7, LcIRF8, and LcIRF10 were more strongly induced by poly (I:C) than the other LcIRFs. Significant induction of all LcIRFs was observed in the spleen, with LcIRF2, LcIRF5, LcIRF6, LcIRF7, LcIRF9, and LcIRF11 reaching their highest levels at 48 h LcIRF3 and LcIRF11 showed a stronger response to poly (I:C) in the spleen than the other LcIRFs. In addition, LcIRF1, LcIRF3, LcIRF7, LcIRF9, LcIRF10, and LcIRF11 were significantly induced by Vibro alginolyticus in both the spleen and the head kidney, with LcIRF1 strongly induced. Thus, LcIRFs exhibited differential inducible expression patterns in response to different stimuli in different tissues, suggesting that LcIRFs have different functions in the regulation of immune responses. Furthermore, overexpression of LcIRF11 activated the promoters of LcIFNc, LcIFNd, and LcIFNh, and differentially induced the expression levels of LcIFNs and IFN-stimulated genes (ISGs). Overexpression of LcIRF11 in epithelioma papulosum cyprinid (EPC) cells inhibited the replication of viral genes after infection of spring viremia of carp virus (SVCV). These data suggested that LcIRF11 may function as a positive regulator in regulating the cellular antiviral response through induction of type I IFN expression. Taken together, the present study reported molecular characterization and expression analysis of 11 IRFs in the large yellow croaker, and investigated the role of LcIRF11 in the antiviral response, which laid a good foundation for further study on the evolution and functional characterization of fish IRFs.

The combination between two well-studied bioactive compounds melatonin and salicylic acid with proper modifications unexpectedly creates a sharp pair of “scissors” cutting off the vicious connection between inflammation and cancer by targeting a key contributor Signal Transducers and Activators of Transcription 3 (STAT3) in the two pathological processes. A representative compound P-3 with IC50 values on each tested cell line ranging from 7.37 to 18.62 μM among the designed melatonin derivatives is equipped with the ability of curbing inflammation-promoting cancer by down-regulating the expression, activation and nuclear translocation of STAT3, breaking the feedforward loop of STAT3 activation by decreasing the expression of pro-tumorigenic cytokines, and inducing cell apoptosis through ROS triggered Cyto-c/Caspase-3 pathway. This study suggests that the melatonin derivative P-3 is likely to become a promising chemical structure for developing the novel anti-cancer agents taking effect through hindering the mutual-promoting processes between inflammation and cancer.

Considerable differences in molecular characteristics have been defined between non-smoker and smokers in patients with lung adenocarcinoma (LUAD), yet study of open chromatin patterns associated with LUAD progression caused by smoking is still lacking. Here, we constructed a novel network based on correlations between each ATAC-seq peak from TCGA data using our previously developed algorithm. Subsequently, principal component analysis was performed on LUAD samples with retained peaks filtered by the correlation network and pathway analysis was conducted for potential pathways identification. Results were verified in an independent dataset from primary LUAD samples. We identified a set of peaks that clearly differentiated long-term from short-term smokers in LUAD patients and also significantly associated with overall survival of these patients. We then investigated the gene set related to those peaks and found that the comprising genes are strongly associated with LUAD development, such as B3GNT3, ACTN4 and CLDN3. They are consistent with the important roles for the associated pathways in LUAD oncogenesis induced by smoking, including glycosphingolipid biosynthesis and tight junction pathways.In summary, our study may provide valuable insights on exploration of ATAC-seq peaks and on smoking-related LUAD carcinogenesis from a perspective of open chromatin changes.

How ocean currents shape fungal transport, dispersal and more broadly fungal biogeography remains poorly understood. The East China Sea (ECS) is a complex and dynamic habitat with different water masses blending microbial communities. The internal transcribed spacer 2 region of fungal rDNA was analysed in water and sediment samples directly collected from the coastal (CWM), Kuroshio (KSWM), Taiwan warm (TWM) and the shelf mixed water mass (MWM), coupled with hydrographic properties measurements, to determine how ocean currents impact the fungal community composition. Almost 9k fungal operational taxonomic units (OTUs) spanning six phyla, 25 known classes, 102 orders and 694 genera were obtained. The typical terrestrial and freshwater fungal genus, Byssochlamys, was dominant in the CWM, while increasing abundance of a specific OTU affiliated with Aspergillus was revealed from coastal to open ocean water masses (TWM and KSWM). Compared with water samples, sediment harboured an increased diversity with distinct fungal communities. The proximity of the Yangtze and Qiantang estuaries homogenizes the surface water and sediment communities. A significant influence of ocean currents on community structure was found, which is believed to reduce proportionally the variation explained by environmental parameters at the scale of the total water masses. Dissolved oxygen and depth were identified as the major parameters structuring the fungal community. Our results indicate that passive fungal dispersal driven by ocean currents and river run-off, in conjunction with the distinct hydrographic conditions of individual water masses, shapes the fungal community composition and distribution pattern in the ECS.

Sesquiterpenoids with diverse skeleton types are regarded as potential lead compounds in pharmacological and other applications. Herein, we report the discovery of two new cadinane-type sesquiterpenoids, paecilacadinol A (1) and B (2); two new drimane-type sesquiterpenoids, ustusol D (3) and ustusol E (4); and six known analogs (5–10) from the endophytic fungus Paecilomyces sp. TE-540, enriching the structural diversity of naturally occurring sesquiterpenoids. Their planar structures were determined on the basis of detailed interpretation of 1D and 2D NMR spectroscopy and HRESIMS data, while their stereochemical structures were established by X-ray crystallographic analyses for 1 and 3–8 and theoretical calculations for 2. Notably, compounds 1 and 2 represent novel examples of cadinane-type sesquiterpenoids with ether bonds formed by intramolecular dehydration. Compounds 5 and 6 showed moderate activities against acetylcholinesterase (AChE), with IC50 values of 43.02 ± 6.01 and 35.97 ± 2.12 μM, respectively. Docking analysis predicted that 5 bound well in the catalytic pocket of AChE via hydrophobic interactions with Trp84, Gly117, Ser122, and Tyr121 residues, while 6 was located with Asp72 and Ser122 residues.

Candida albicans is an opportunistic human pathogen, and its pathogenicity is associated with hyphal formation. Previous studies have shown that at neutral-to-alkaline pH, hyphal growth is dependent on the Rim101 pathway whose activation requires Snf7, a member of the ESCRT system. In this work, we described the purification and characterization of the C. albicans Vps4, an AAA ATPase required for recycling of the ESCRTs. Its role on hyphal growth has been investigated. Our data suggest deletion of Vps4 decreases overall hyphal growth at pH 7 and increases the growth of multiple hyphae induced by serum, which indicates that the ESCRTs may make a Rim101-independent contribution to hyphal growth. Furthermore, DBeQ, an inhibitor of the AAA ATPase p97, was shown to inhibit the ATPase activity of Vps4 with an IC50 of about 11.5 μM. To a less degree, it also inhibits hyphal growth. Our work may provide a new strategy to control C. albicans infection.

Root-knot nematodes are obligate parasites that feed on plant roots and cause serious crop losses worldwide. Bacillus species (Bacilliaceae) can produce nematicidal metabolites and have shown good potential for biological control of nematodes. In this study, Bacillus mycoides strain R2 isolated from rhizosphere soil of tomato plants exhibited high nematicidal activity against the free-living nematode Caenorhabditis elegans and the root-knot nematode Meloidogyne incognita. In a pot experiment, control efficiency of B. mycoides R2 on M. incognita was as high as 90.94%. The nematicidal compound was isolated and identified as styrene. The median lethal concentration of styrene against M. incognita was 4.55 μg/ml (m/v). The volatile styrene caused avoidance and killed nematodes primarily by the olfactory neuron and G protein signal pathway. C. elegans detected styrene with the AWB neuron; the signal was then transmitted to the downstream G protein coupled receptors CHE-3, DOP-3, and STR-2. Then signal activated G protein GPA-3 and GPA-7. The signal was then transmitted to ion channels (CNGs channel and TRPV channel), causing calcium ion internal flow and a stress response towards the increased concentration of intracellular calcium. Styrene should be registered as a nematode repellent and biocontrol agent for protection of crops against root-knot nematode attack.

Human monocytic ehrlichiosis (HME) is caused by an obligatory intracellular bacterium, E. chaffeensis, and is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. The mechanisms through which E. chaffeensis invades and establishes an intracellular niche are not well understood but are dependent on secreted ehrlichial effector proteins. The significance of this study is in addressing how intracellular pathogens, particularly those with small genomes such as Ehrlichia, exploit a limited number of secreted effector proteins such as tandem repeat proteins (TRPs) to manipulate complex eukaryotes and to regulate host cell processes through molecular pathogen-host interplay. The results of our studies highlight the broader role of ehrlichial TRPs in promoting infection and help define the mechanisms through which obligately intracellular bacteria modulate host cell function for survival., Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes. The mechanisms involved in E. chaffeensis infection of the host cell and evasion of host defenses are not fully defined, but a subset of type 1 secreted tandem repeat protein (TRP) effectors play important roles. Recently, we determined molecular interactions of TRP120, TRP47, and TRP32 with the eukaryotic host cell. In this investigation, we used yeast two-hybrid analysis to reveal that another E. chaffeensis tandem repeat protein, TRP75, interacts with a diverse group of human proteins associated with organismal and tissue homeostasis, multiple metabolic processes and regulation, response to reactive oxygen species, signal transduction, and protein modifications. Thirteen identified host target proteins associated with actin cytoskeleton reorganization or apoptosis were examined in detail and confirmed to interact with TRP75 at different levels as determined by coimmunoprecipitation assays. These protein interactions were visualized by immunofluorescence confocal microscopy during infection and colocalized with Ehrlichia morulae with different intensities. Moreover, small interfering RNAs (siRNAs) (n = 86) were used to knock down identified TRP75-interacting host proteins separately, and their influence on ehrlichial infection was investigated by real-time quantitative PCR (qPCR). Knockdown of 74/86 (86%) TRP75 target proteins had a significant negative effect on ehrlichial infection. The results of this study further support the idea of a role of Ehrlichia TRPs as effectors that interact with a complex array of host proteins to promote ehrlichial infection. IMPORTANCE Human monocytic ehrlichiosis (HME) is caused by an obligatory intracellular bacterium, E. chaffeensis, and is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. The mechanisms through which E. chaffeensis invades and establishes an intracellular niche are not well understood but are dependent on secreted ehrlichial effector proteins. The significance of this study is in addressing how intracellular pathogens, particularly those with small genomes such as Ehrlichia, exploit a limited number of secreted effector proteins such as tandem repeat proteins (TRPs) to manipulate complex eukaryotes and to regulate host cell processes through molecular pathogen-host interplay. The results of our studies highlight the broader role of ehrlichial TRPs in promoting infection and help define the mechanisms through which obligately intracellular bacteria modulate host cell function for survival.

Ehrlichia chaffeensis is an obligately intracellular Gram-negative bacterium that selectively infects mononuclear phagocytes. We recently reported that E. chaffeensis utilizes a type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors and demonstrated that these effectors interact with a functionally diverse array of host proteins. By way of these interactions, TRP effectors modulate host cell functions; however, the molecular basis of these interactions and their roles in ehrlichial pathobiology are not well defined. In this study, we describe the first bacterial protein posttranslational modification (PTM) by the small ubiquitin-like modifier (SUMO). The E. chaffeensis T1S effector TRP120 is conjugated to SUMO at a carboxy-terminal canonical consensus SUMO conjugation motif in vitro and in human cells. In human cells, TRP120 was selectively conjugated with SUMO2/3 isoforms. Disruption of TRP120 SUMOylation perturbed interactions with known host proteins, through predicted SUMO interaction motif-dependent and -independent mechanisms. E. chaffeensis infection did not result in dramatic changes in the global host SUMOylated protein profile, but a robust colocalization of predominately SUMO1 with ehrlichial inclusions was observed. Inhibiting the SUMO pathway with a small-molecule inhibitor had a significant impact on E. chaffeensis replication and recruitment of the TRP120-interacting protein polycomb group ring finger protein 5 (PCGF5) to the inclusion, indicating that the SUMO pathway is critical for intracellular survival. This study reveals the novel exploitation of the SUMO pathway by Ehrlichia , which facilitates effector-eukaryote interactions necessary to usurp the host and create a permissive intracellular niche.

In multicellular organisms, autophagy is induced as an innate defense mechanism. Notably, the obligate intracellular bacterium Ehrlichia chaffeensis resides in early endosome-like vacuoles and circumvents lysosomal fusion through an unknown mechanism, thereby avoiding destruction in the autophagolysosome. In this report, we reveal that Wnt signaling plays a crucial role in inhibition of lysosomal fusion and autolysosomal destruction of ehrlichiae. During early infection, autophagosomes fuse with ehrlichial vacuoles to form an amphisome indicated by the presence of autophagy markers such as LC3 (microtubule-associated protein 1 light chain 3), Beclin-1, and p62. LC3 colocalized with ehrlichial morulae on days 1, 2, and 3 postinfection, and increased LC3II levels were detected during infection, reaching a maximal level on day 3. Ehrlichial vacuoles did not colocalize with the lysosomal marker LAMP2, and lysosomes were redistributed and dramatically reduced in level in the infected cells. An inhibitor specific for the Wnt receptor signaling component Dishevelled induced lysosomal fusion with ehrlichial inclusions corresponding to p62 degradation and promoted transcription factor EB (TFEB) nuclear localization. E. chaffeensis infection activated the phosphatidylinositol 3-kinase (PI3K)–Akt–mTOR (mechanistic target of rapamycin) pathway, and activation was induced by three ehrlichial tandem repeat protein (TRP) effectors, with TRP120 inducing the strongest activation. Moreover, induction of glycogen synthase kinase-3 (GSK3) performed using a Wnt inhibitor and small interfering RNA (siRNA) knockdown of critical components of PI3K-GSK3-mTOR signaling decreased ehrlichial survival. This report reveals Ehrlichia exploitation of the evolutionarily conserved Wnt pathway to inhibit autolysosome generation, thereby leading to evasion of this important innate immune defense mechanism.

Ehrlichia chaffeensis infects mononuclear phagocytes and survives intracellularly by exploiting host cell processes to evade host defenses. The mechanisms involved are not fully defined, but appear to rely largely on a subset of tandem repeat proteins (TRP) effectors. E. chaffeensis TRPs are type 1 secreted effectors that interact with a functionally diverse group of host cell targets associated with various biological processes. In this study, we investigated the influence of TRP host target proteins on ehrlichial infection by RNA interference. In total, 138 TRP-interacting host proteins identified by yeast two-hybrid were targeted by siRNA and the infection level determined by real-time qPCR. Knockdown of 124 (89%) TRP target proteins had significant influence on infection either by inhibiting (85%) or promoting (15%) ehrlichial infection. Notably, knockdown of 18 host proteins which interacted with TRP120 promoted the infection, suggesting that these targets may be degraded to promote infection. Host proteins that interact with TRPs are involved in cellular processes, including cell signaling, vesicle trafficking and intracellular transport, transcriptional regulation, metabolism, protein posttranslational modification, and apoptosis. Selected host targets were examined by immunofluorescent microscopy during infection and were found to localize with the morulae, or in the host cell cytoplasm adjacent to morulae. This study confirms that the majority of host proteins known to interact with TRP effectors influence infection and further extends the current knowledge that E. chaffeensis TRPs participate in a complex array of host protein interactions in order to reprogram the host cell and promote intracellular survival.

Determining the complex relationships between diseases, polymorphisms in human genes and environmental factors is challenging. Multifactor dimensionality reduction (MDR) has been proven to be capable of effectively detecting the statistical patterns of epistasis, although classification accuracy is required for this approach. The imbalanced dataset can cause seriously negative effects on classification accuracy. Moreover, MDR methods cannot quantitatively assess the disease risk of genotype combinations. Hence, we introduce a novel weighted risk score-based multifactor dimensionality reduction (WRSMDR) method that uses the Bayesian posterior probability of polymorphism combinations as a new quantitative measure of disease risk. First, we compared the WRSMDR to the MDR method in simulated datasets. Our results showed that the WRSMDR method had reasonable power to identify high-order gene-gene interactions, and it was more effective than MDR at detecting four-locus models. Moreover, WRSMDR reveals more information regarding the effect of genotype combination on the disease risk, and the result was easier to determine and apply than with MDR. Finally, we applied WRSMDR to a nasopharyngeal carcinoma (NPC) case-control study and identified a statistically significant high-order interaction among three polymorphisms: rs2860580, rs11865086 and rs2305806.

Increased expression of phospholipid transfer protein (PLTP) was observed in the brains of Alzheimer's disease (AD) patients; however, the role of PLTP in the progress of AD is still poorly understood. The objective of this study was to evaluate the effect of PLTP deficiency on the recognition and metabolism of amyloid-β (Aβ) in mice. We performed the Morris water maze to determine the learning and memory capabilities of 50-week age wild type mice (WT, n = 12) and PLTP knockout mice (PLTP-/-, n = 12). The levels of Aβ and amyloid-β protein precursor (AβPP) were examined by ELISA and western blot, respectively. The levels and activity of β- and γ-secretases were determined by western blot and activity assay kit, respectively. Morris water maze results showed that PLTP deficiency significantly impaired recognition compared with WT mice. Levels of Aβ42 in the cortex and hippocampus was significantly increased, yet the levels of Aβ40 in the cortex was decreased in PLTP-/- compared with WT mice. No typical senile plaques were found in the WT or PLTP-/- mice. AβPP expression and β-secretase activity were both significantly increased in PLTP-/- mice. Moreover, PLTP deficiency increased the expression of γ-secretase catalytic units and decreased the content of apolipoprotein E. Therefore we concluded that PLTP deficiency impaired cognition and aggravated AD by enhancing the generation of Aβ in the cortex of old mice.

Sphingosine-1-phosphate (S1P) is an amphiphilic signaling molecule, which is enriched in functional high density lipoprotein (HDL) and shows arterial protection. The distribution of S1P is changed with increased plasma phospholipid transfer protein (PLTP) activity and impaired HDL function in patients with coronary heart diseases. Therefore, we hypothesized that PLTP might transfer S1P among cells or lipoproteins. We found that plasma S1P contents were decreased by 60.1 % in PLTP knockout mice (PLTP−/−, N = 5) compared with their wild type littermates (WT, N = 5) (151.70 ± 38.59 vs. 379.32 ± 59.90 nmol/l, P

Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival., IMPORTANCE E. chaffeensis is an obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection and avoids innate host defenses are not understood, but functionally relevant host-pathogen interactions with type 1 secreted TRP effectors are essential for the ehrlichial cellular reprogramming strategy. This study provides further insight into the molecular strategies used by obligately intracellular pathogens such as E. chaffeensis, which have small genomes and a limited number of effector proteins and exploit evolutionarily conserved host cell programs such as Notch signaling to promote infection and intracellular survival.

Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival.

Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of β-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, β-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca 2+ and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival.

Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions of E. chaffeensis tandem repeat proteins 47 and 120 (TRP47 and -120) and the eukaryotic host cell have been described. In this investigation, yeast two-hybrid analysis demonstrated that an E. chaffeensis type 1 secretion system substrate, TRP32, interacts with a diverse group of human proteins associated with major biological processes of the host cell, including protein synthesis, trafficking, degradation, immune signaling, cell signaling, iron metabolism, and apoptosis. Eight target proteins, including translation elongation factor 1 alpha 1 (EF1A1), deleted in azoospermia (DAZ)-associated protein 2 (DAZAP2), ferritin light polypeptide (FTL), CD63, CD14, proteasome subunit beta type 1 (PSMB1), ring finger and CCCH-type domain 1 (RC3H1), and tumor protein p53-inducible protein 11 (TP53I11) interacted with TRP32 as determined by coimmunoprecipitation assays, colocalization with TRP32 in HeLa and THP-1 cells, and/or RNA interference. Interactions between TRP32 and host targets localized to the E. chaffeensis morulae or in the host cell cytoplasm adjacent to morulae. Common or closely related interacting partners of E. chaffeensis TRP32, TRP47, and TRP120 demonstrate a molecular convergence on common cellular processes and molecular cross talk between Ehrlichia TRPs and host targets. These findings further support the role of TRPs as effectors that promote intracellular survival.

The optimal conditions of production of biopolymer by the culture of Bacillus sp.were examined, using molasses wastewater to replace glucose as carbon source and energy source in the culture medium. Results showed that the COD concentration in molasses wastewater favorable for the growth of the Bacillus sp.was 4500 mg•L-1, and inoculum size of 6%(v/v), 28◦C, initial pH 7.0 and shaking speed of 160 r•min-1, under the optimal culture conditions, the highest flocculating activity achieved for Kaolin suspension was 97.5% and 4.0 g biomass /L broth was obtained. The Bacillus sp. biomass was effective in flocculating some soluble reactive dyes in aqueous solution, reactive Light-Yellow K-4G and reactive Turquoise Blue KN-G with a decolorization efficiency of 98.5 and 92.5%, respectively, using 20 mL of the flocculant in 500 mL of 100 mg•L-1 dye solution.

The ethanolic extract of propolis (EEP) is beneficial in increasing high density lipoprotein (HDL) cholesterol (HDL-C) and diminishing risks of atherosclerosis. In this study, we examined the effects of EEP on reverse cholesterol transport in mice. (3)H -cholesterol laden macrophage was injected intraperitoneally into mice fed by gastric gavage with EEP. Plasma lipid level was determined and (3)H-cholesterol was traced in plasma, liver and feces. The effects of EEP on ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) and scavenger receptor BI (SR-BI) in mice liver and in cultured cells were also investigated. EEP administration led to a significant increase in HDL-C and peritoneal macrophage-original (3)H-cholesterol in plasma, liver and feces. Liver protein expressions of ABCA1 and ABCG1 were increased but SR-B1 was not. In vitro experiments with HepG2 and Raw264.7 cell lines confirmed the above results. The finding of these studies shows that EEP-enhanced reverse cholesterol transport may have resulted from EEP stimulated plasma HDL level and hepatic ABCA1 and ABCG1 expression.

In the present study we reported the feasibility of extracellular biopolymer(PF01) produced by Pseudomonas fluorescence as an alternative biosorbent to remove Cu(II) ions from environmental wastewater. The ability of PF01 to remove the Cu(II) ions was investigated by using batch biosorption procedure. The effects such as pH, dosage of biosorbent, Cu(II) initial concentration and sorbate–sorbent contact time on the adsorption were studied. The pseudo-second order kinetics and Langmuir isotherm models were noted be fit to the experimental data. The maximum Cu(II) adsorption capacity was 78.99 mg/g at pH 5.0 , at 28°C. The carboxyl , hydroxyl and amino groups of the PF01 were involved in chemical interaction with the Cu(II) ions depicted by Fourier transform infrared spectroscopic (FTIR). The study points to the potential of new use of extracellular biopolymer PF01 as an effective biosorbent for the removal of Cu(II) from environmental wastewater.

Our previous studies have demonstrated that mice with reduced or absent serotonin transporter (SERT+/- and SERT-/- mice, respectively) are more sensitive to stress relative to their SERT normal littermates (SERT+/+ mice). The aim of the present study was to test the hypothesis that the hypothalamic-pituitary-adrenal (HPA) axis and its feedback regulation are impaired in these mice. The function and gene expression of several components in the HPA axis and its feedback regulation in SERT+/+, +/( and -/- mice were studied under basal (non-stressed) and stressed conditions. The results showed that (1) under basal conditions, corticotrophin-releasing factor (CRF) mRNA levels in the paraventricular nucleus (PVN) of the hypothalamus was lower in both SERT+/( and (/( mice relative to SERT+/+ mice; (2) an increased response to CRF challenge was found in SERT(/( mice, suggesting that the function of CRF type 1 receptors (CRF R1) in the pituitary is increased. Consistent with these findings, (125)I-sauvagine (a CRF receptor antagonist) binding revealed an increased density of CRF R1 in the pituitary of SERT(/( under basal conditions. These data suggest that CRF R1 in the pituitary of SERT(/( mice is up-regulated. However, in the pituitary of SERT+/( mice, the function of CRF R1 was not changed and the density of CRF R1 was reduced relative to SERT+/+ mice; and (3) the expression of the glucocorticoid receptor (GR) in the hypothalamus, pituitary and adrenal cortex was significantly reduced in SERT+/( and (/( mice in comparison with SERT+/+ mice under basal conditions. Consistent with these findings, the corticosterone response to dexamethasone was blunted in SERT(/( mice relative to SERT+/+ and +/( mice. Furthermore, stress induces a rapid increase of the GR expression in the hypothalamus of SERT+/( and (/( mice relative to their basal levels. Together, the present results demonstrated that the HPA axis and its feedback regulation are altered in SERT knockout mice, which could account for the increased sensitivity to stress in these mice.

Aim Chitosan-oligosaccharides (COS) treatment showed lipid lowering effects in rats and reverse cholesterol transport (RCT) promotion in mice, suggested that COS might be a potential atheroprotective material. In this study, we investigated the effects of COS treatment on atherosclerosis (AS) in apolipoprotein E deficient mice (apoE-/-). Methods After feeding high fat (HF) diet for 12 weeks with the gastric gavages administration of COS or vehicle, respectively, the mice were sacrificed for the assessment of atherosclerosis, plaque stability, and the mechanism investigation. Results Cholesterol and TG in non-high density lipoprotein (non-HDL) fractions were reduced dramatically in COS groups. The COS treatment decreased the lesion areas of aortic enface, plaque areas in aortic root, and increased plaque stability in apoE-/-. Furthermore, the COS treatment significantly enhanced the expression of liver low density lipoprotein receptor (LDL-R), scavenger receptor BI (SR-BI) as well as the expression of macrophage SR-BI and ATP binding cassette transporter A1(ABCA1). We also found that the COS treatment did not affect the plasma lipid level in LDL-R deficient mice and cholesterol absorption in wild type mice. Conclusions COS treatment attenuated AS and decreased plasma non-HDL level in apoE-/-, and the potential mechanism might be involved with enhanced expression of hepatic LDL-R and SR-BI, and macrophage ABCA1.

In invertebrates, C-type lectin plays an important role in innate immunity by mediating the recognition of pathogens to host cells and clearing microinvaders. A few C-type lectins have been identified from shrimps, but none of their gene or protein sequences is known to date. In this paper, a C-type lectin (named PmLec) specific for bacterial lipopolysaccharide was purified from the serum of the shrimp Penaeus monodon. The binding of PmLec to lipopolysaccharide was mainly mediated through the O-antigen. PmLec had a strong hemagglutinating and bacterial-agglutinating activity as well as an opsonic effect that enhances hemocyte phagocytosis. The PmLec cDNA sequence was obtained from the cDNA library of P. monodon by polymerase chain reaction with the degenerated primer designed according to the amino-terminal residue sequence of purified PmLec. A 546-bp open reading frame was found to encode a putative protein comprising 182 amino acids and containing a preceding signal peptide of 17 amino acids. A C-type lectin domain existed in PmLec, but no glycosylation site was found. The recombinant PmLec protein expressed in Escherichia coli also showed the same agglutinating activity and opsonic effect as that of the native protein. This is the first report of a lectin cDNA from the shrimp. PmLec functions as a pattern-recognition protein and an opsonin in the shrimp, and it provides a clue to elucidate the role of lectin in the innate immunity of aquatic invertebrates at the molecular level.

Objective— Phospholipid transfer protein (PLTP) accelerates the development of atherosclerosis in mouse models. We examined the role of PLTP in atherosclerotic plaque stability. Approach and Results— We prepared apolipoprotein E and PLTP double-knockout (PLTP −/− ApoE −/− ) mice. PLTP deficiency significantly decreased lesion size and reduced monocyte/macrophage infiltration, as well as macrophage apoptosis in lesion areas. Moreover, it increased fibrous content in plaques, which suggests that PLTP may affect atherosclerotic plaque stability. Importantly, PLTP overexpression mediated by adenovirus had the reverse effect. It promoted the accumulation of reactive oxygen species in macrophages, which could lead to cell apoptosis and increased the production of inflammatory cytokines and chemokines. PLTP overexpression could promote receptor-interacting protein 3 recruitment of macrophages in cytoplasm, which could induce reactive oxygen species, thus inducing atherogenesis. Conclusions— PLTP plays an important role in modulating the stability of atherosclerotic plaques. The receptor-interacting protein 3- reactive oxygen species signal pathway could be involved in this PLTP-mediated process.

Low level of immunity for SARS-CoV among well healthcare workers reinforces the need for infection control measures in hospitals to prevent epidemics., To determine the prevalence of inapparent infection with severe acute respiratory syndrome (SARS) among healthcare workers, we performed a serosurvey to test for immunoglobulin (Ig) G antibodies to the SARS coronavirus (SARS-CoV) among 1,147 healthcare workers in 3 hospitals that admitted SARS patients in mid-May 2003. Among them were 90 healthcare workers with SARS. As a reference group, 709 healthcare workers who worked in 2 hospitals that never admitted any SARS patients were similarly tested. The seroprevalence rate was 88.9% (80/90) for healthcare workers with SARS and 1.4% (15/1,057) for healthcare workers who were apparently healthy. The seroprevalence in the reference group was 0.4% (3/709). These findings suggest that inapparent infection is uncommon. Low level of immunity among unaffected healthcare workers reinforces the need for adequate personal protection and other infection control measures in hospitals to prevent future epidemics.

Diseases caused by viruses especially by white spot syndrome virus (WSSV) are the greatest challenge to worldwide shrimp aquaculture. The innate immunity of shrimp has attracted extensive attention, but no factor involved in the virus resistance has been reported. Here we report for the first time the identification of an antiviral gene from shrimp Penaeus monodon. A differential cDNA (designated as PmAV) cloned from virus-resistant shrimp P. monodon by differential display (DD) was found to have an open reading frame (ORF) encoding a 170 amino acid peptide with a C-type lectin-like domain (CTLD). The PmAV gene was expressed in Escherichia coli and the protein was purified. Recombinant PmAV protein displayed a strong antiviral activity in inhibiting virus-induced cytopathic effect in fish cell in vitro. Moreover, native PmAV protein was isolated from shrimp hemolymph by immuno-affinity chromatography and confirmed by Western blot. No agglutination activity was observed both in recombinant and native PmAV protein. Immunohistological study showed that PmAV protein was located mainly in the cytoplasm, and not bound to the shrimp WSSV. It implies that the antiviral mechanism of PmAV protein is not by inhibiting the attachment of virus to target host cell. The discovery of PmAV gene might provide a clue to elucidate the innate immunity of marine invertebrates and would be helpful to shrimp viral disease control.

Ehrlichia chaffeensis is an obligately intracellular gram negative bacterium with a small genome that thrives in mammalian mononuclear phagocytes by exploiting eukaryotic processes. Herein, we discuss the latest findings on moonlighting tandem repeat protein effectors and their secretion mechanisms, and novel molecular interkingdom interactions that provide insight into the intracellular pathobiology of ehrlichiae.

Humoral immune mechanisms are an important component of protective immunity to Ehrlichia species. However, the molecular basis of antibody mediated immunity is not completely defined, and the role of most molecularly characterized major immunoreactive proteins is unknown. In previous studies, we mapped major species-specific continuous epitopes in three surface exposed and secreted tandem repeat proteins (TRP32, TRP47 and TRP120). In this study, we report that protection is provided by antibodies against these molecularly defined TRP epitopes using in vitro and in vivo models. Protection was demonstrated in vitro after prophylactic and therapeutic administration of epitope-specific anti-TRP antibodies, suggesting that the protective mechanisms involve extracellular and intracellular antibody-mediated effects. In vivo passive transfer of individual epitope-specific TRP sera significantly reduced the ehrlichial load and splenomegaly, and protected mice against lethal infection. Moreover, the combination of antibodies to all three TRPs provided enhanced reduction in ehrlichial load similar to that of Ehrlichia chaffeensis immune sera. IgG1 was the predominant antibody isotype in the epitope-specific TRP mouse sera. These results demonstrate that antibodies against linear epitopes in TRP32, TRP47 and TRP120 are protective during E. chaffeensis infection and involves extracellular and intracellular antibody-mediated mechanisms.

The Cu(II) biosorption characteristics of Bacillus cereus B-11 biomass was examined as a function of initial pH, contact time, and initial metal ion concentration. The pseudo-second-order kinetic model was found to be well-suited for the entire adsorption process of Cu(II) on B-11. Langmuir and Freundlich models were applied to describe the biosorption isotherm of the copper ions by B-11 biomass. Langmuir model fitted the equilibrium data was better than the Freundlich isotherm. The maximum binding capacity of Cu(II) according to the Langmuir isotherm was 88.14 mg/g at pH 6.5 and shaker speed was 150 rpm at 27°C and for 60 min. The present study indicates that B-11 biomass may be used as an inexpensive, effective, and easily cultivable biosorbent for the removal of Cu(II) ions from environmental and industrial wastewater.

Ehrlichia chaffeensis is an obligately intracellular bacterium that modulates host cell gene transcription in the mononuclear phagocyte, but the host gene targets and mechanisms involved in transcriptional modulation are not well-defined. In this study, we identified a novel tandem repeat DNA-binding domain in the E. chaffeensis 120-kDa tandem repeat protein (TRP120) that directly binds host cell DNA. TRP120 was observed by immunofluorescent microscopy in the nucleus of E. chaffeensis -infected host cells and was detected in nuclear extracts by Western immunoblotting with TRP120-specific antibody. The TRP120 binding sites and associated host cell target genes were identified using high-throughput deep sequencing (Illumina) of immunoprecipitated DNA (chromatin immunoprecipitation and high-throughput DNA sequencing). Multiple em motif elicitation (MEME) analysis of the most highly enriched TRP120-bound sequences revealed a G+C-rich DNA motif, and recombinant TRP120 specifically bound synthetic oligonucleotides containing the motif. TRP120 target gene binding sites were mapped most frequently to intersecting regions (intron/exon; 49%) but were also identified in upstream regulatory regions (25%) and downstream locations (26%). Genes targeted by TRP120 were most frequently associated with transcriptional regulation, signal transduction, and apoptosis. TRP120 targeted inflammatory chemokine genes, CCL2, CCL20, and CXCL11, which were strongly upregulated during E. chaffeensis infection and were also upregulated by direct transfection with recombinant TRP120. This study reveals that TRP120 is a novel DNA-binding protein that is involved in a host gene transcriptional regulation strategy.

Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions between E. chaffeensis 47-kDa tandem repeat (TR) protein (TRP47) and the eukaryotic host cell have been described. In this investigation, yeast ( Saccharomyces cerevisiae ) two-hybrid analysis demonstrated that E. chaffeensis -secreted tandem repeat protein 120 (TRP120) interacts with a diverse group of host cell proteins associated with major biological processes, including transcription and regulation, cell signaling, protein trafficking, and actin cytoskeleton organization. Twelve target proteins with the highest frequency of interaction with TRP120 were confirmed by cotransformation in yeast. Host targets, including human immunoglobulin lambda locus (IGL), cytochrome c oxidase subunit II (COX2), Golgi-associated gamma adaptin ear-containing ARF binding protein 1 (GGA1), polycomb group ring finger 5 (PCGF5), actin gamma 1 (ACTG1), and unc-13 homolog D (UNC13D; Caenorhabditis elegans ), colocalized strongly with TRP120 in HeLa cells and with E. chaffeensis dense-cored morulae and areas adjacent to morulae in the host cytoplasm. The TR domain of TRP120 interacted only with PCGF5, indicating that distinct TRP120 domains contribute to specific host target interactions and that multiple domains are required to reconstitute TRP120 interactions with other host targets. Three previously defined molecular interactions between TRP47 and host proteins, PCGF5, IGLL1, and CAP1, were also associated with TRP120, demonstrating that molecular cross talk occurs between Ehrlichia TRPs and host targets. These findings further support the role of TRPs as effectors that reprogram the host cell.

It is well known that chitosan has beneficial lipid-regulating effects, but it remains unknown whether chitosan oligosaccharide (COS), the chitosan degradation product, has the same lipid benefits. High-fat-diet-fed Wistar rats were administrated with COS by gastric gavage for three weeks. The effects of COS on lipids, lipoprotein components and lipid metabolism related protein activities were investigated. Plasma lipids level assays by an enzyme method showed that COS decreased triglyceride (TG) by 29–31%, and increased high-density lipoprotein (HDL) cholesterol by 8–11%, but did not affect low-density lipoprotein (LDL) cholesterol. Lipid distribution analysis through fast protein liquid chromatography indicated that COS significantly decreased TG content distributed in very-low-density lipoprotein (VLDL)/LDL fractions but increased cholesterol content in HDL fractions. Apolipoprotein analysis through plasma ultracentrifugation and sodium dodecyl sulfate polyacrylamide gel electrophoresis displayed that COS decreased apolipoprotein B-100 of LDL and increased apolipoprotein E of LDL and apolipoprotein B-100 of VLDL, but did not change apoA-I content of HDL particles. Lipoprotein formation associated protein determination showed that COS also increased plasma activity of lecithin cholesterol acyl transferase but not phospholipid transfer protein. The present study suggests that COS may play a beneficial role in plasma lipid regulation of rats with dyslipidemia induced by high-fat diet. The COS-decreased VLDL/LDL TG and -enhanced HDL cholesterol may be related to the upregulated activity of lecithin cholesterol acyl transferase.

Hydrogen (dihydrogen; H(2)) has an antiatherosclerotic effect in apolipoprotein (apo) E knockout mice. The goals of this study were to further characterize the effects of H(2) on the content, composition, and biological activities of plasma lipoproteins in golden hamsters. Plasma analysis by enzymatic method and fast protein liquid chromatography showed that 4-week intraperitoneal injection of hydrogen-saturated saline remarkably decreased plasma total cholesterol and low-density lipoprotein (LDL) cholesterol levels in high-fat diet-fed hamsters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of apolipoproteins from ultracentrifugally isolated plasma lipoproteins revealed a marked decrease of apo B100 and apo B48 in LDL. A profound decrease of apo E level in very low-density lipoprotein was also observed. Besides, we determined the functional quality of high-density lipoprotein (HDL) particles isolated from H(2)-treated and control mice. H(2) significantly improved HDL functionality assessed in 2 independent ways, namely, (1) stimulation of cholesterol efflux from macrophage foam cells by measuring HDL-induced [(3)H]cholesterol efflux and (2) protection against LDL oxidation as a measure of Cu(2+)-induced thiobarbituric acid reactive substances formation. Administration of hydrogen-saturated saline decreases plasma LDL cholesterol and apo B levels and improves hyperlipidemia-injured HDL functions, including the capacity of enhancing cellular cholesterol efflux and playing antioxidative properties, in high-fat diet-fed hamsters.

The present study was undertaken to evaluate the feasibility of Pseudomonas alcaligenes biomass for the removal of strontium ions from aqueous solutions. Batch experiments were performed to study the adsorption of strontium on pH, Pseudomonas alcaligenes biomass adsorbent with respect to initial Sr(II) concentration, contact time and biomass dose. The experimental data were modeled by Langmuir and Freundlich isotherm models. Langmuir model resulted in the best fit of the adsorption data. The maximum adsorption capacity for Sr(II) was 67.35 mg/g (pH 6.0 and 5 g•L-1 biomass dose). Kinetics of adsorption followed second-order rate equations. The FTIR results of Pseudomonas alcaligenes biomass showed that biomass has different functional groups and these functional groups are able to react with metal ion in aqueous solution. The results of the present study suggest that Pseudomonas alcaligenes biomass can be used beneficially in treating industrial effluents containing heavy metal ions.

Recently, major species-specific antibody epitopes in three immunoreactive tandem repeat proteins (TRPs) of Ehrlichia chaffeensis , TRP32, TRP47, and TRP120, have been identified and molecularly characterized within tandem repeat (TR) regions. In this study, we mapped the major immunodeterminants of the E. chaffeensis 200-kDa ankyrin protein (Ank200) and the minor immunodeterminants in the N- and C-terminal regions of E. chaffeensis TRP47. Major antibody epitopes of Ank200 were localized to four polypeptide regions (18-mer, 20-mer, 20-mer, and 21-mer, respectively) in terminal acidic domains, which reacted with antibodies in sera from human monocytotropic ehrlichiosis (HME) patients and an E. chaffeensis -infected dog. Two minor epitope-containing regions were identified in the N terminus and the C terminus of TRP47. The sensitivities and specificities of synthetic peptides representing these and other well-defined major immunodeterminants of E. chaffeensis were determined by enzyme-linked immunosorbent assay (ELISA). Thirty-one HME patient serum samples that had detectable E. chaffeensis antibodies (titers from 64 to 8,192) by indirect fluorescent-antibody assay (IFA) were tested. All 31 serum samples reacted with at least one E. chaffeensis peptide, 30 (96.8%) with TRP120 peptides, 27 (87.1%) with TRP32 peptides, 24 (77.4%) with TRP47 peptides, 19 (61.3%) with Ank200 peptides, and 28 (90.3%) with recombinant TRP120-TR protein. A mixture of the two most sensitive peptides from TRP120 and TRP32 did not provide enhanced analytical sensitivity compared to that provided by TRP120 alone. Our results demonstrate that the TRP120 peptide can be utilized for development of standardized sensitive point-of-care and reference laboratory immunodiagnostics for HME. This is the first study to compare analysis of molecularly defined major antibody epitopes with IFA for diagnosis of HME.

Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing protein orthologs, including p120/p140, which elicit strong antibody responses. The TR regions of these protein orthologs are immunoreactive, but the molecular characteristics of the p120/p140 epitopes have not been determined. In this study, the immunodeterminants of the E. chaffeensis p120 and E. canis p140 were identified and molecularly defined. Major antibody epitope-containing regions of both p120 and p140 were localized to the TR regions, which reacted strongly by Western immunoblotting with antibodies in sera from E. chaffeensis -infected dogs or patients and E. canis- infected dogs, respectively. Single continuous species-specific major epitopes within the E. chaffeensis p120 and E. canis p140 TRs were mapped to homologous surface-exposed glutamate/aspartate-rich regions (19 to 22 amino acids). In addition, minor cross-reactive epitopes were localized to homologous N- and C-terminal regions of p120 and p140. Furthermore, although the native and recombinant p120 and p140 proteins exhibited higher-than-predicted molecular masses, posttranslational modifications were not present on abnormally migrating p120 and p140 TR recombinant proteins as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The extent of knowledge regarding the diversity of globally distributedEhrlichia canisstrains has been limited to information gained from a few evolutionarily conserved genes. In this study,E. canisstrains from the United States (strain Jake [US]), Brazil (strain São Paulo [BR]), and Israel (strain 611 [IS] and Ranana [IS-R]) were used to examine the antigenic and genetic diversities of four well-characterized major immunoreactive protein genes/proteins. gp36 and gp200 were the most divergent genes, and nucleotide substitutions in the gp36 tandem repeat region of the IS strain, but not the IS-R strain, resulted in two amino acid differences (S→P and P→T) in each nine-amino-acid repeat (epitope-containing region). DNA sequences of gp19 and gp140 were completely conserved in the US and BR strains, but differences were found in the Israeli strains, including two fewer tandem repeats in gp140 and a single amino acid substitution in gp19 from the IS strain.E. caniswhole-cell lysates from each isolate were examined by Western immunoblotting using sera from naturally infected dogs from each country, and four major immunoreactive proteins (gp19, gp36, gp140, and gp200) were identified in each strain using protein-specific antisera. The US and BR strains exhibited highly conserved immunoreactive protein profiles, while some differences were identified in the IS strain. Sera from naturally infected Israeli dogs confirmed gene sequencing information, which demonstrated two distinctE. canisstrains, defined by the gp36 gene. Conversely, gp19 was strongly reactive and present in allE. canisisolates. gp140 and gp200 were also present in all strains, although gp140 in the IS strain had two fewer tandem repeats and exhibited a smaller mass.

Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing proteins that elicit strong host immune responses and are associated with host-pathogen interactions. In a previous study, we molecularly characterized a highly conserved 19-kDa major immunoreactive protein (gp19) of E. canis and identified the corresponding TR-containing ortholog variable-length PCR target (VLPT) protein in E. chaffeensis . In this study, the native 32-kDa VLPT protein was identified and the immunodeterminants defined in order to further understand the molecular basis of the host immune response to E. chaffeensis . Synthetic and/or recombinant polypeptides corresponding to various regions of VLPT were used to localize major antibody epitopes to the TR-containing region. Major antibody epitopes were identified in three nonidentical repeats (R2, R3, and R4), which reacted strongly with antibodies in sera from an E. chaffeensis -infected dog and human monocytotropic ehrlichiosis patients. VLPT-R3 and VLPT-R2 reacted most strongly with antibody, and the epitope was further localized to a nearly identical proximal 17-amino-acid region common between these repeats that was species specific. The epitope in R4 was distinct from that of R2 and R3 and was found to have conformational dependence. VLPT was detected in supernatants from infected cells, indicating that the protein was secreted. VLPT was localized on both reticulate and dense-core cells, and it was found extracellularly in the morula fibrillar matrix and associated with the morula membrane.

Hemocyanin, the respiratory protein of arthropods and mollusks, was found to function against bacterial and fungal invasion. In this study, we show that this protein complex could delay the infection of white spot syndrome virus (WSSV) in vivo and cloned two hemocyanin subunits genes (PjHcL and PjHcY) from the shrimp Penaeus japonicus. Further investigation of their transcriptional regulation during viral invasion showed that in contrast to subunit PjHcY, PjHcL expression could be strongly induced by WSSV infection. These findings not only reveal an important role of hemocyanin in shrimp antiviral defense but also suggest a possible discrepancy between the two subunits in shrimp innate immunity.

Most research on the adaptation of thermophiles is focused on their adaptation to heat stress; only a few studies are focused on their cold adaptation. In this report, the thermophilic bacterium Rhodothermus sp. XMH10 was examined to gain a better understanding of gene expression in response to low temperature. Random arbitrarily primed polymerase chain reaction (RAP-PCR) was used to isolate and identify differentially expressed genes of bacteria grown at 45 degrees C (lowest) compared to those at 75 degrees C (optimal). Fifty-three differential cDNA fragments in total were isolated. Among them, 35 different cDNAs were analyzed by Northern blot, and 17 were confirmed to be differentially expressed at the transcriptional levels. These genes reflected a profile of differential expression and were involved in many physiological processes such as metabolism, cell membrane alterations, and regulatory adaptive response; most of them have never been previously reported. This study provides some new information on the adaptation of thermophilic bacteria to environmental temperature stress.