Triple-gene deletion for osteocalcin significantly impairs the alignment of hydroxyapatite crystals and collagen in mice.

Osteocalcin (Ocn), also known as bone Gla protein, is synthesized by osteoblasts and thought to regulate energy metabolism, testosterone synthesis and brain development. However, its function in bone is not fully understood. Mice have three Ocn genes: Bglap, Bglap2 and Bglap3. Due to the long span of these genes in the mouse genome and the low expression of Bglap3 in bone, researchers commonly use Bglap and Bglap2 knockout mice to investigate the function of Ocn. However, it is unclear whether Bglap3 has any compensatory mechanisms when Bglap and Bglap2 are knocked out. Considering the controversy surrounding the role of Ocn in bone, we constructed an Ocn-deficient mouse model by knocking out all three genes (Ocn-/-) and analyzed bone quality by Raman spectroscopy (RS), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and MicroCT (µCT). The RS test showed that the alignment of hydroxyapatite crystals and collagen fibers was significantly poorer in Ocn-/- mice than in wild-type (WT) mice. Ocn deficiency resulted in a looser surface structure of bone particles and a larger gap area proportion. FTIR analysis showed few differences in bone mineral index between WT and Ocn-/- mice, while µCT analysis showed no significant difference in cortical and trabecular regions. However, under tail-suspension simulating bone loss condition, the disorder of hydroxyapatite and collagen fiber alignment in Ocn-/- mice led to more obvious changes in bone mineral composition. Collectively, our results revealed that Ocn is necessary for regulating the alignment of minerals parallel to collagen fibrils.

A Microelectrode Array Modified by PtNPs/PB Nanocomposites Used for the Detection and Analysis of Glucose-Sensitive Neurons under Different Blood Glucose States.

Hypoglycemia state damages the organism, and glucose-excited and glucose-inhibited neurons from the ventral medial hypothalamus can regulate this state. Therefore, it is crucial to understand the functional mechanism between blood glucose and electrophysiology of glucose-excited and glucose-inhibited neurons. To better detect and analyze this mechanism, a PtNPs/PB nanomaterials modified 32-channel microelectrode array with low impedance (21.91 ± 6.80 kΩ), slight phase delay (-12.7° ± 2.7°), high double layer capacitance (0.606 µF), and biocompatibility was developed to realize in vivo real-time detection of the electrophysiology activities of glucose-excited and glucose-inhibited neurons. The phase-locking level of some glucose-inhibited neurons elevated during fasting (low blood glucose state) and showed theta rhythms after glucose injection (high blood glucose state). With an independent oscillating ability, glucose-inhibited neurons can provide an essential indicator to prevent severe hypoglycemia. The results reveal a mechanism for glucose-sensitive neurons to respond to blood glucose. Some glucose-inhibited neurons can integrate glucose information input and convert it into theta oscillating or phase lock output. It helps in enhancing the interaction between neurons and glucose. Therefore, the research can provide a basis for further controlling blood glucose by modulating the characteristics of neuronal electrophysiology. This helps reduce the damage of organisms under energy-limiting conditions, such as prolonged manned spaceflight or metabolic disorders.

Hepatic metabolite responses to 4-day complete fasting and subsequent refeeding in rats.

Background: Fasting has been widely used to improve various metabolic diseases in humans. Adaptive fasting is necessary for metabolic adaptation during prolonged fasting, which could overcome the great advantages of short-term fasting. The liver is the main organ responsible for energy metabolism and metabolic homeostasis. To date, we lack literature that describes the physiologically relevant adaptations of the liver during prolonged fasting and refeeding. For that reason, this study aims to evaluate the response of the liver of Sprague-Dawley (SD) rats to prolonged fasting and refeeding. Methods: Sixty-six male SD rats were divided into the fasting groups, which were fasted for 0, 4, 8, 12, 24, 48, 72, or 96 h, and the refeeding groups, which were refed for 1, 3, or 6 days after 96 h of fasting. Serum glucose, TG, FFA, ß-hydroxybutyrate, insulin, glucagon, leptin, adiponectin and FGF21 levels were assessed. The glucose content, PEPCK activity, TG concentration and FFA content were measured in liver tissue, and the expression of genes involved in gluconeogenesis (PEPCK and G6Pase), ketogenesis (PPARα, CPT-1a and HMGCS2) and the protein expression of nutrient-sensing signaling molecules (AMPK, mTOR and SIRT1) were determined by RT-qPCR and western blotting, respectively. Results: Fasting significantly decreased the body weight, which was totally recovered to baseline after 3 days of refeeding. A 4-day fast triggered an energy metabolic substrate shift from glucose to ketones and caused serum hormone changes and changes in the protein expression levels of nutrient-sensing signaling molecules. Glycogenolysis served as the primary fuel source during the first 24 h of fasting, while gluconeogenesis supplied the most glucose thereafter. Serum FFA concentrations increased significantly with 48 h of fasting. Serum FFAs partly caused high serum ß-hydroxybutyrate levels, which became an important energy source with the prolongation of the fasting duration. One day of refeeding quickly reversed the energy substrate switch. Nutrient-sensing signaling molecules (AMPK and SIRT1 but not mTOR signaling) were highly expressed at the beginning of fasting (in the first 4 h). Serum insulin and leptin decreased with fasting initiation, and serum glucagon increased, but adiponectin and FGF21 showed no significant changes. Herein, we depicted in detail the timing of the metabolic response and adaptation of the liver to a 4-day water-only fast and subsequent refeeding in rats, which provides helpful support for the design of safe prolonged and intermittent fasting regimens.

Effects of 10-Day Complete Fasting on Physiological Homeostasis, Nutrition and Health Markers in Male Adults.

Fasting shows great potential in preventing chronic diseases and has to be surmounted under some extraordinary circumstances. This study aimed to investigate the safety, time effects of metabolic homeostasis and health indexes during prolonged fasting. Thirteen participants were recruited to conduct a 10-day complete fasting (CF) in a controlled health research building under medical supervision including 3-day Baseline (BL), 10-day CF, 4-day calorie restriction (CR) and 5-day full recovery (FR). Body healthy status was assessed by surveying pulse, blood pressure, body weight (BW), blood glucose and ketones, body composition and nutritional and biochemistry indexes at different times. BW declined about 7.28 kg (-9.8%) after 10-day CF, accompanied by increased pulse and decreased systolic blood pressure, but there were no changes to the myocardial enzymogram. Body composition analysis showed fat mass was constantly lost, but lean mass could recover after CR. The energy substrate switch from glucose to ketone occurred and formed a stable dynamic balance between 3-6 days of CF. The lipid metabolism presented increased total cholesterol, LDL-C, ApoA1 and almost no changes to TG and HDL-C. Prolonged CF did not influence liver function, but induced a slight decrease of kidney function. The interesting results came from the marked increase of lipid-soluble vitamins and a significant decrease of sodium and chlorine. Adults could well tol-erate a 10-day CF. A new metabolic homeostasis was achieved. No vitamins but NaCl supplement should be considered. These findings provide evidence to design a new fasting strategy for clinical practice.

Undercarboxylated osteocalcin inhibits the early differentiation of osteoclast mediated by Gprc6a.

Osteocalcin (OCN) was the most abundant noncollagen protein and considered as an endocrine factor. However, the functions of Undercarboxylated osteocalcin (ucOCN) on osteoclast and bone resorption are not well understood. In the present study, preosteoclast RAW264.7 cells and bone marrow mononuclear cells (BMMs) were treated with ucOCN purified from prokaryotic bacteria. Our results showed that ucOCN attenuated the proliferation of RAW264.7 cells with a concentration dependant manner by MTS assay. Scrape wounding assay revealed the decreased motility of RAW264.7 cells after ucOCN treatment. RT-qPCR results manifested the inhibitory effects of ucOCN on the expression of osteoclastic marker genes in RAW264.7 cells during inducing differentiation of RANKL. It was also observed that ucOCN inhibited the formation of multinucleated cells from RAW264.7 cells and BMMs detected by TRAP staining. The number and area of bone resorb pits were also decreased after treatment with ucOCN during their osteoclast induction by toluidine blue staining. The formation and integrity of the osteoclast actin ring were impaired by ucOCN by immunofluorescent staining. Time dependant treatment of ucOCN during osteoclastic induction demonstrated the inhibitory effects mainly occurred at the early stage of osteoclastogenesis. Signaling analysis of luciferase activity of the CRE or SRE reporter and ERK1/2 phosphorylation showed the selective inhibitor or siRNA of Gprc6a (a presumptive ucOCN receptor) could attenuate the promotion of ucOCN on CRE-luciferase activity. Taken together, we provided the first evidence that ucOCN had negative effects on the early differentiation and bone resorption of osteoclasts via Gprc6a.

Potential Diagnostic Value of the Peripheral Blood Mononuclear Cell Transcriptome From Cattle With Bovine Tuberculosis.

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis. During early-stage infection, M. bovis-infected cattle shed mycobacteria through nasal secretions, which can be detected via nested-polymerase chain reaction (PCR) experiments. Little research has focused on immune responses in nested PCR-positive (bTB PCR-P) or nested PCR-negative (bTB PCR-N) M. bovis-infected cattle. Here, we investigated the transcriptomes of peripheral blood mononuclear cells (PBMCs), with or without stimulation by purified protein derivative of bovine tuberculin (PPD-B), among bTB PCR-P, bTB PCR-N, and healthy cattle using RNA-Seq. We also explored the potential value of PBMC transcripts as novel biomarkers for diagnosing bTB. Numerous differentially expressed genes were identified following pair-wise comparison of different groups, with or without PPD-B stimulation (adjusted p < 0.05). Compared with healthy cattle, bTB PCR-P, and bTB PCR-N cattle shared 5 significantly dysregulated biological pathways, including Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, Hematopoietic cell lineage, Osteoclast differentiation and HTLV-I infection. Notably, dysregulated biological pathways of bTB PCR-P and bTB PCR-N cattle were associated with cell death and phagocytosis, respectively. Lymphotoxin alpha and interleukin-8 could potentially differentiate M. bovis-infected and healthy cattle upon stimulation with PPD-B, with area-under-the-curve (AUC) values of 0.9991 and 0.9343, respectively. B cell lymphoma 2 and chitinase 3-like 1 might enable differentiation between bTB PCR-P and bTB PCR-N upon stimulation with PPD-B, with AUC values of 0.9100 and 0.8893, respectively. Thus, the PBMC transcriptome revealed the immune responses in M. bovis-infected cattle (bTB PCR-P and bTB PCR-N) and may provide a novel sight in bTB diagnosis.

Ketotifen fumarate attenuates feline gingivitis related with gingival microenvironment modulation.

Gingivitis is evidenced by inflammation of the free gingiva, and still reversible. If left untreated, it may then progress to periodontitis. In the present study, the therapeutical effect of ketotifen fumarate on gingivitis was explored. Domestic cats with varying degrees of gingivitis naturally were enrolled in this study. Subgroups of animals were treated twice daily for one week with or without ketotifen fumarate (5 mg/kg). Effects of ketotifen fumarate were measured on gingival index, cells accumulation, mediators release, receptor-ligand interaction, oxidative stress, MAPK and NF-κB pathways, epithelial barrier and apoptosis. Ketotifen fumarate attenuated the initiation and progression of gingivitis, inhibited the infiltrations of mast cells, B lymphocytes, T lymphocytes, macrophages, neutrophils and eosinophils as well as the release of IgE, ß-hexosaminidase, tryptase, chymase, TNF-α, IL-4, and IL-13, influenced endothelial cells, fibroblasts and epithelial cells proliferation and apoptosis, and induced Th2 cells polarization, where ketotifen fumarate also might affect their interactions. Ketotifen fumarate reduced the oxidative stress, and inhibited NF-κB and p38 MAPK related with mast cells and macrophages accumulation. Ketotifen fumarate improved the aberrant expression of ZO-1 and inhibits the following apoptosis. On the other hand, these cells and mediators augmented functional attributes of them involving SCF/c-Kit, α4ß7/VCAM-1 and IL-8/IL-8RB interactions, thus creating a positive feedback loop to perpetuate gingivitis, where an inflammation microenvironment was modeled. Our results showed a previously unexplored therapeutic potential of ketotifen fumarate for gingivitis and further suggest that, in addition to biofilms, targeting inflammation microenvironment could be new strategy for the treatment of gingivitis/periodontitis.

Foot-and-mouth disease virus non-structural protein 2B negatively regulates the RLR-mediated IFN-ß induction.

Foot-and-mouth disease virus (FMDV) is the causative agent of Foot-and-mouth disease (FMD), which is an acute and highly contagious disease affecting pigs, cattle and other cloven-hoofed animals. Several studies have shown that FMDV has evolved multiple strategies to evade the host innate immune response, but the underlying mechanisms for immune evasion are still not fully understood. In the current research, we have demonstrated that FMDV utilizes its non-structural protein 2B to sabotage the host immune response. Over-expression of the FMDV 2B inhibited Poly(I:C)-induced or SeV-triggered up-regulation of IFN-ß, IL-6 as well as ISG15. When HEK293T cells were transfected with FMDV 2B, the phosphorylation of TBK1 and IRF3 was inhibited. Co-immunoprecipitation and pull-down experiments indicated that FMDV 2B protein could interact with host RIG-I and MDA5. Moreover, FMDV 2B also inhibited the expression of the RIG-I and MDA5. Thus, FMDV 2B negatively regulates the RLR-mediated IFN-ß induction by targeting RIG-I and MDA5.

Genomic sequence and virulence of a novel NADC30-like porcine reproductive and respiratory syndrome virus isolate from the Hebei province of China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), which results in immense economic losses in the swine industry. Outbreaks of disease caused by NADC30-like PRRSV are of great concern in China. Here, a novel variant, NADC30-like PRRSV strain HB17A, was analyzed and its pathogenicity in pigs was examined. The full-length genome sequence of HB17A shared 83.6-95.1% nucleotide similarity with NADC30-like and NADC30 PRRSV without any gene insertions, but with a unique 2-amino acid deletion in Nsp2. A phylogenetic analysis showed that HB17A clustered with NADC30 strains. Different degrees of variation in the signal peptide, transmembrane region (TM), primary neutralizing epitope (PNE), non-neutral epitopes, and N-glycosylation sites were observed in GP5. Challenge experiments showed that HB17A infection resulted in persistent fever, moderate respiratory clinical signs, low levels of viremia and viral loads in serum, and mild gross and microscopic lung lesions. Moreover, IFN-γ, IL-6, and IL-10 cytokine levels were significantly elevated in serum, but the levels of IFN-α and IL-2 were similar to those of the negative controls. HB17A was less pathogenic but was secreted longer in nasal discharge than HP-PRRSV FZ06A. Our findings indicate that HB17A is a novel NADC30-like strain with certain deletions and mutations but with no evidence of genomic recombination. This strain exhibits intermediate virulence in pigs. This research will be help define the evolutionary characteristics of Chinese NADC30-like PRRSV.

Limitations of Using IL-17A and IFN-γ-Induced Protein 10 to Detect Bovine Tuberculosis.

Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in M. bovis-infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB.

Genomic characterization and pathogenic study of two porcine reproductive and respiratory syndrome viruses with different virulence in Fujian, China.

Two strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in 2006 and 2016 and designated as FZ06A and FZ16A, respectively. Inoculation experiments showed that FZ06A caused 100% morbidity and 60% mortality, while FZ16A caused 100% morbidity without death. By using genomic sequence and phylogenetic analyses, close relationships between a Chinese highly pathogenic PRRSV strain and the FZ06A and FZ16A strains were observed. Based on the achieved results, multiple genomic variations in Nsp2, a unique N-glycosylation site (N³³â†’K³³), and a K151 amino acid (AA) substitution for virulence in the GP5 of FZ16A were detected; except the 30 AA deletion in the Nsp2-coding region. Inoculation experiments were conducted and weaker virulence of FZ16A than FZ06A was observed. Based on our results, a 30 AA deletion in the Nsp2-coding region is an unreliable genomic indicator of a high virulence PRRSV strain. The Nsp2 and GP5 differences, in addition to the virulence difference between these two highly pathogenic PRRSV strains, have the potential to be used to establish a basis for further study of PRRSV virulence determinants and to provide data useful in the development of vaccines against this economically devastating disease.

Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA) vaccine candidates for the surveillance and eradication of PPR.