Oral Administered Particulate Yeast-Derived Glucan Promotes Hepatitis B Virus Clearance in a Hydrodynamic Injection Mouse Model

Hepatitis B virus (HBV) persistent infection is associated with ineffective immune response for the clearance of virus. Immunomodulators represent an important class of therapeutics, which potentially could be beneficial for the treatment of HBV infection. The particulate yeast-derived glucan (PYDG) has been shown to enhance the innate and adaptive immune responses. We therefore, assessed the efficacy of PYDG in enhancing HBV specific immune responses by employing the hydrodynamic injection-based (HDI) HBV transfection mouse model. Mice were intragatric administered PYDG daily for 9 weeks post pAAV/HBV1.2 hydrodynamic injection. PYDG treatment significantly promoted HBV DNA clearance and production of HBsAb compared to control mice. PYDG treatment resulted in recruitment of macrophages, dendritic cells (DCs) and effector T cells to the liver microenvironment, accompanied by a significantly augmented DCs maturation and HBV-specific IFN-γ and TNF-α production by T cell. In addition, enhanced production of Th1 cytokines in liver tissue interstitial fluid (TIF) was associated with PYDG administration. Live imaging showed the accumulation of PYDG in the mouse liver. Our results demonstrate that PYDG treatment significantly enhances HBV-specific Th1 immune responses, accompanied by clearance of HBV DNA, and therefore holds promise for further development of therapeutics against chronic hepatitis B.     

Genotyping of Human Brucella melitensis Biovar 3 Isolated from Shanxi Province in China by MLVA16 and HOOF

Background

Brucellosis presents a significant economic burden for China because it causes reproductive failure in host species and chronic health problems in humans. These problems can involve multiple organs. Brucellosis is highly endemic in Shanxi Province China. Molecular typing would be very useful to epidemiological surveillance. The purpose of this study was to assess the diversity of Brucella melitensis strains for epidemiological surveillance. Historical monitoring data suggest that Brucella melitensis biovar 3 is the predominant strain associated with the epidemic of brucellosis in Shanxi Province.

Methods/Principal Findings

Multiple-locus variable-number repeat analysis (MLVA-16) and hypervariable octameric oligonucleotide fingerprinting (HOOF-print) were used to type a human-hosted Brucella melitensis population (81 strains). Sixty-two MLVA genotypes (discriminatory index: 0.99) were detected, and they had a genetic similarity coefficient ranging from 84.9% to 100%. Eighty strains of the population belonged to the eastern Mediterranean group with panel 1 genotypes 42 (79 strains) and 43 (1 strain). A new panel 1 genotype was found in this study. It was named 114 MLVAorsay genotype and it showed similarity to the two isolates from Guangdong in a previous study. Brucella melitensis is distributed throughout Shanxi Province, and like samples from Inner Mongolia, the eastern Mediterranean genotype 42 was the main epidemic strain (97%). The HOOF-printing showed a higher diversity than MLVA-16 with a genetic similarity coefficient ranging from 56.8% to 100%.

Conclusions

According to the MLVA-16 and HOOF-printing results, both methods could be used for the epidemiological surveillance of brucellosis. A new genotype was found in both Shanxi and Guangdong Provinces. In areas with brucellosis, the MLVA-16 scheme is very important for tracing cases back to their origins during outbreak investigations. It may facilitate the expansion and eradication of the disease.     

Mineral Elements of Subtropical Tree Seedlings in Response to Elevated Carbon Dioxide and Nitrogen Addition

Mineral elements in plants have been strongly affected by increased atmospheric carbon dioxide (CO₂) concentrations and nitrogen (N) deposition due to human activities. However, such understanding is largely limited to N and phosphorus in grassland. Using open-top chambers, we examined the concentrations of potassium (K), calcium (Ca), magnesium (Mg), aluminum (Al), copper (Cu) and manganese (Mn) in the leaves and roots of the seedlings of five subtropical tree species in response to elevated CO₂ (ca. 700 μmol CO₂ mol^-1) and N addition (100 kg N ha^-1 yr^-1) from 2005 to 2009. These mineral elements in the roots responded more strongly to elevated CO₂ and N addition than those in the leaves. Elevated CO₂ did not consistently decrease the concentrations of plant mineral elements, with increases in K, Al, Cu and Mn in some tree species. N addition decreased K and had no influence on Cu in the five tree species. Given the shifts in plant mineral elements, Schima superba and Castanopsis hystrix were less responsive to elevated CO₂ and N addition alone, respectively. Our results indicate that plant stoichiometry would be altered by increasing CO₂ and N deposition, and K would likely become a limiting nutrient under increasing N deposition in subtropics.     

Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.     

Spag17 Deficiency Results in Skeletal Malformations and Bone Abnormalities

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.     

A Comprehensive Alanine-Scanning Mutagenesis Study Reveals Roles for Salt Bridges in the Structure and Activity of Pseudomonas aeruginosa Elastase

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (k _cat/K _m), half-lives (t _1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.     

Activity Prediction and Molecular Mechanism of Bovine Blood Derived Angiotensin I-Converting Enzyme Inhibitory Peptides

Development of angiotensin I-converting enzyme (ACE, EC 3.4.15.1) inhibitory peptides from food protein is under extensive research as alternative for the prevention of hypertension. However, it is difficult to identify peptides released from food sources. To accelerate the progress of peptide identification, a three layer back propagation neural network model was established to predict the ACE-inhibitory activity of pentapeptides derived from bovine hemoglobin by simulated enzyme digestion. The pentapeptide WTQRF has the best predicted value with experimental IC₅₀ 23.93 μM. The potential molecular mechanism of the WTQRF / ACE interaction was investigated by flexible docking.     

MiR-506 Suppresses Tumor Proliferation and Invasion by Targeting FOXQ1 in Nasopharyngeal Carcinoma

MiRNAs are small noncoding RNAs that play important roles in various biological processes including tumorigenesis. However, little is known about the expression and function of miR-506 in nasopharyngeal carcinoma (NPC). In this study, we showed that miR-506 was downregulated in nasopharyngeal carcinoma (NPC) cell lines and tissues. Ectopic expression of miR-506 dramatically suppressed cell proliferation, colony formation and invasion. Moreover, we identified the Forkhead box Q1 (FOXQ1) gene as a novel direct target of miR-506. MiR-506 exerts its tumor suppressor function through inhibition of the FOXQ1, which was involved in tumor metastasis and proliferation in various cancers. Furthermore, the expression of FOXQ1 is up-regulated in NPC cell lines and tissues. Taken together, our results indicate that miR-506 functions as a tumor suppressor miRNA in NPC and that its suppressive effects are mediated chiefly by repressing FOXQ1 expression.     

Olfactory Dysfunction and Neurotransmitter Disturbance in Olfactory Bulb of Transgenic Mice Expressing Human A53T Mutant α-Synuclein

Parkinson disease is a multi-system neurodegenerative disease characterized by both motor and non-motor symptoms. Hyposmia is one of the early non-motor symptoms occurring in more than 90% of Parkinson disease cases, which can precede motor symptoms even several years. Up to now, the relationship between hyposmia and Parkinson disease remains elusive. Lack of proper animal models of hyposmia restricts the investigation. In this study we assessed olfactory function in Prp-A53T-α-synuclein transgenic (αSyn^A53T) mice which had been reported to show age-dependent motor impairments and intracytoplasmic inclusions. We also examined cholinergic and dopaminergic systems in olfactory bulb of αSyn^A53T mice by immunofluorescent staining, enzyme linked immunosorbent assay and western blot. We found that compared to wild type littermates, αSyn^A53T mice at 6 months or older displayed a deficit of odor discrimination and odor detection. No significant changes were found in olfactory memory and odor habituation. Furthermore compared to wildtype littermates, in olfactory bulb of αSyn^A53T mice at 10 months old we detected a marked decrease of cholinergic neurons in mitral cell layer and a decrease of acetylcholinesterase activity, while dopaminergic neurons were found increased in glomerular layer, accompanied with an increase of tyrosine hydroxylase protein. Our studies indicate that αSyn^A53T mice have olfactory dysfunction before motor deficits occur, and the cholinergic and dopaminergic disturbance might be responsible for the Parkinson disease-related olfactory dysfunction.