Nitisinone attenuates cartilage degeneration and subchondral osteoclastogenesis in osteoarthritis and concomitantly inhibits the cGAS/STING/NF-κB pathway.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degeneration and subchondral bone remodelling. Currently, conservative treatment strategies cannot effectively alleviate the progression of OA. In this study, we used computer network analysis to show that Nitisinone (NTBC) is closely related to extracellular matrix degradation in OA and mainly interferes with the TNF-α signaling pathway. NTBC is an orphan drug used to treat hereditary type I tyrosinemia by altering phenylalanine/tyrosine metabolic flow. In this study, we found that NTBC effectively reduced chondrocyte inflammation and extracellular matrix degradation induced by TNF-α. Mechanistically, NTBC inhibited the cGAS/STING signaling pathway and reduced activation of the STING-dependent NF-κB pathway to alleviate inflammation. In addition, NTBC inhibited osteoclastogenesis and delayed the occurrence of subchondral bone remodelling. In mice with ACLT-induced osteoarthritis, intra-articular injection of NTBC significantly reduced cartilage degradation and subchondral bone remodelling. NTBC showed impressive therapeutic efficacy as a potential pharmaceutical intervention for the treatment of OA.

Carnitine functions as an enhancer of NRF2 to inhibit osteoclastogenesis via regulating macrophage polarization in osteoporosis.

Osteoporosis, which manifests as reduced bone mass and deteriorated bone quality, is common in the elderly population. It is characterized by persistent elevation of macrophage-associated inflammation and active osteoclast bone resorption. Currently, the roles of intracellular metabolism in regulating these processes remain unclear. In this study, we initially performed bioinformatics analysis and observed a significant increase in the proportion of M1 macrophages in bone marrow with aging. Further metabolomics analysis demonstrated a notable reduction in the expression of carnitine metabolites in aged macrophages, while carnitine was not detected in osteoclasts. During the differentiation process, osteoclasts took up carnitine synthesized by macrophages to regulate their own activity. Mechanistically, carnitine enhanced the function of Nrf2 by inhibiting the Keap1-Nrf2 interaction, reducing the proteasome-dependent ubiquitination and degradation of Nrf2. In silico molecular ligand docking analysis of the interaction between carnitine and Keap1 showed that carnitine binds to Keap1 to stabilize Nrf2 and enhance its function. In this study, we found that the decrease in carnitine levels in aging macrophages causes overactivation of osteoclasts, ultimately leading to osteoporosis. A decrease in serum carnitine levels in patients with osteoporosis was found to have good diagnostic and predictive value. Moreover, supplementation with carnitine was shown to be effective in the treatment of osteoporosis.

Effect of an Acinetobacter pittobacter on low-density polyethylene.

Low-density polyethylene (LDPE) is a major cause of persistent and long-term environmental pollution. In this study, we report the successful isolation of an efficient LDPE degrading bacterial strain from the wastewater sediment of an agricultural land film recycling plant and identified and designated it as Acinetobacter sp. LW-1, respectively. The surface analyses including scanning electron microscopy (SEM) and atomic force microscopy (AFM) of LW-1-treated samples revealed the existence of appreciable pits and cavities on the facet of PE film, while the formation of carbonyl group was also verified by XPS and FTIR. Moreover, water contact angle assay substantiated the chemical transformation of the LDPE film from hydrophobic to hydrophilic transition after treatment with LW-1. In addition, co-incubation of LDPE film and Acinetobacter sp. LW-1 at 35 °C for 90 days significantly decreased the weight of LDPE film with an optimal weight loss of 15 ± 0.85%. This work enriches the LDPE degradation bacterial library and shows the tremendous potential of Acinetobacter sp. LW-1 to be used in the degradation of LDPE waste.

Biodegradability of polyethylene mulch film by Bacillus paramycoides.

Discarded polyethylene (PE) mulch film has led to persistent agricultural pollution. Biodegradation of plastic waste is considered as a promising solution that can potentially overcome environmental and economic problems. In this study, a novel bacterium (Bacillus paramycoides) was isolated from a waste mulch recycling plant and showed an extraordinary ability to customize polyethylene film. It was observed by scanning electron microscopy that a large number of pits and wrinkle cracks existed on the polyethylene, indicating that the strain used PE film as the sole carbon source. Meanwhile, the loss of weight of the film was tested continuously, and approximately 12% of the initial weight of the film was found to be lost within 45 days after coincubation with TW-2. The surface hydrophobicity of the polyethylene film decreased while the surface tension increased from 9.755 to 31.013. Fourier transform infrared (FTIR) analysis indicated that absorption peaks near 1740 cm-1 and 2760 cm-1 were attributed to the stretching vibrations of aldehyde and carboxyl groups, respectively, suggesting that hydrophilic groups were produced. This was also confirmed by XPS spectroscopy analysis. X-ray diffraction (XRD) analysis also showed that the relative crystallinity decreased from 33% to 11.51%. In addition, GPC analysis showed that the molecular weight decreased, while the proportion of low molecular weight fragments increased. These results strongly indicated that the PE film was able to be degraded to some extent by the strain. Finally, a new biodegradable mechanism for polyethylene was proposed.

[Effect of ultrasonic scaling on micro-leakage of all-ceramic crowns].

PURPOSE: To investigate the effect of ultrasonic scaling on micro-leakage of all-ceramic crowns. METHODS: 48 first premolars of the similar shape and size were collected and prepared by one attending doctor. Two groups were randomly divided (zirconia crown group and glass ceramic crown group, with 24 in each group), and each group was randomly divided into two subgroups (resin cement group and resin modified glass cement subgroup, n=6). After the crowns were bonded and solidified for 24 h, 3 samples were randomly selected from each subgroup for ultrasonic scaling for 60 s, and the other 3 samples were used as the negative control group. After all samples were soaked in 5% fuchsin solution at room temperature for 48 h, buccal and lingual incisions were made, and double-blind scoring was performed for micro-leakage at the crown edge of each sample. SPSS 17.0 software package was used for statistical analysis. RESULTS: There were significant differences between the negative group and the ultrasonic group in micro-leakage of crowns which were cemented with resin modified glass cement(P<0.05), but there was no significant difference between the negative group and the ultrasonic group in micro-leakage of crowns which were cemented with resin cement(P>0.05). When the same adhesive was used, there was significant difference in microleakage between the zirconia crown and the glass-ceramic crown, whether ultrasonic supragingival scaling was performed or not(P<0.05). CONCLUSIONS: Ultrasonic scaling had significant effect on micro-leakage of crowns which were cemented with resin modified glass cement, and but had no significant effect on micro-leakage of crowns which were cemented with resin cement.

Alternative isoforms of BmYki have different transcriptional co-activator activity in the silkworm, Bombyx mori.

Yorki (Yki), a transcriptional co-activator that is a key component of the Hippo pathway, induces the transcription of a number of targets that promote cell proliferation and survival. Bombyx mori Yki3 (BmYki3), with 445 amino acid residues, facilitates cell migration and cell division, and enlarges cultured cell and wing disc size. In this study, cellular localization, transcriptional co-activator activity, cell migration, cell cycle, and cell size were characterized in alternative isoforms of BmYki. BmYki1 and BmYki3 are mainly located in the cytoplasm and nucleus, respectively, while, BmYki2 is located in both the cytoplasm and nucleus. The mutation BmYki1S97A (S97mutated to A) was transported from the cytoplasm to nucleus. Cell migration, cell cycle, and cell size could be enhanced by BmYki, however, the effect of BmYki1 and BmYki2 on cell proliferation was less compared to BmYki3. Moreover, wing discs could be enlarged by overexpressing BmYki1 or BmYki2 isoforms. Dual-luciferase reporter assay showed that BmYki3 had the highest activity to B. mori ovarian tumor gene. In BmN cells overexpressing one of the BmYki isoforms, expression levels of kibra ortholog (kibra), inhibitor of apoptosis protein (iap), four-jointed (fj), expanded (ex), crumbs (crb) and BMP and activin membrane-bound inhibitor homolog (Bmpr) genes were upregulated, while those of α-catenin (α-cat), decapentaplegic (dpp), serrate (serr) and signal transducer and activator of transcription (stat) genes were down-regulated. There was some difference in the regulation of gene expression between different isoforms. These results suggested that the activity of BmYki isoforms was different in the silkworm.

Cultured cells and wing disc size of silkworm can be controlled by the Hippo pathway.

Hippo signalling represents a cell proliferation and organ-size control pathway. Yorki (Yki), a component of the Hippo pathway, induces the transcription of a number of targets that promote cell proliferation and survival. The functions of Yki have been characterized in Drosophila and mammals, while there are few reports on silkworm, Bombyx mori In the present study, we found that BmYki3 facilitates cell migration and cell division, and enlarges the cultured cell and wing disc size. Co-immunoprecipitation results indicated that BmYki3 may interact with thymosin, E3 ubiquitin-protein ligase, protein kinase ASK1, dedicator of cytokinesis protein 1, calcium-independent phospholipase A2 and beta-spectrin. RNA-seq results indicated that 4444 genes were upregulated and 10 291 genes were downregulated after BmYki3 was overexpressed in the cultured cells. GO annotation indicated that the up/downregulated genes were enriched in 268/382 GO terms (p < 0.01); KEGG analysis showed that the up/downregulated genes were enriched in 49/101 pathways. These findings provided novel information to understand the functions of BmYki3 in a cell proliferation and organ-size control pathway.

Exogenous gene can be expressed by a recombinant Bombyx mori cypovirus.

Bombyx mori cypovirus (BmCPV) is one of the major viral pathogen for silkworm, and the genome of BmCPV is composed of 10 dsRNA segments. As construction system of recombinant BmCPV (rBmCPV) is scanty, researchers achieved little progress in studying gene function of BmCPV in recent decades. Here, 10 recombinant plasmids with a full-length cDNA of viral genome segments S1-S10 containing T7 promoter were constructed. After cotransfecting the BmN cells with the mixture of 10 in vitro-transcribed RNAs, pathological changes were observed. Real-time PCR and Western blot showed viral gene vp1 and structural proteins were expressed. It is found the genome of the rBmCPV is composed of 10 dsRNA segments similar to those of wild-type BmCPV. Moreover, viral particles and polyhedron with virions can be generated in the cotransfected cells and the injected silkworm midguts. These findings confirmed the formation of infective rBmCPV. Additionally, we found viable rBmCPV was generated by cotransfecting the mixture of in vitro-transcribed S1-S9 RNAs into the cultured cells, confirming polh was not essential for BmCPV replication. Moreover, an infectious rBmCPV expressing the DsRed protein was constructed based on this system. Further investigation showed S2 and S7 segments are indispensible for viral proliferation. Our findings demonstrated the construction system of rBmCPV can be utilized for exploring viral replication and pathogenesis, and investigated method for constructing rBmCPV will certainly facilitate developing novel biopesticides and expressing exogenous gene in the midgut of silkworm.

Novel Infection System of Recombinant BmBDV DNA into BmN Cells of Silkworm, Bombyx mori.

Bombyx mori bidensovirus (BmBDV) was previously termed as Bombyx mori densovirus type 2 and later it was reclassified in the new genus bidensovirus of the new family Bidnaviridae. The genome of BmBDV Zhenjiang isolate (BmBDV-Z) consists of two non-homologous single-stranded linear DNA molecules VD1 and VD2 which are encapsidated into separate virion. To investigate the infectivity of BmBDV DNA, recombinant plasmids pGEM-VD1 inserted with VD1 genome were transfected into the BmN cells of silkworm. Structural proteins of BmBDV were detected with Western blot and immunofluorescence assay, which indicates pGEM-VD1 replicated in the transfected BmN cells and viral proteins were also expressed. Through TEM observation, we identified about 20 nm BmBDV-like viral particles, which confirmed that BmBDV can be generated after transfection. Subsequently, a recombinant baculovirus BmBac-VD1 inserted with VD1 genome was constructed. Results of Western blot and immunofluorescence assay indicated that viral structural proteins of BmBDV were expressed in the BmBac-VD1-infected cells. Baculiform and spherical virions were also observed in infected cells by TEM, and two kinds of virions were separated. However, results of molecular biological detection revealed that infectious sequence from BmBac-VD1 was packaged within spherical virion. Therefore, we suggested that vector inserted with BmBDV genomic DNA showed infectivity, and BmBDV-like viral particles packaging recombinant DNA can be produced in the cultured BmN cells. Outcome of our current research provided not only a new method of infection to explore the gene function of BmBDV in vitro but also a protocol to facilitate development of more effective new-type pesticides.

Exogenous gene can be integrated into Nosema bombycis genome by mediating with a non-transposon vector.

Nosema bombycis, a microsporidium, is a pathogen of pebrine disease of silkworms, and its genomic DNA sequences had been determined. Thus far, the research of gene functions of microsporidium including N. bombycis cannot be performed with gain/loss of function. In the present study, we targeted to construct transgenic N. bombycis. Therefore, hemocytes of the infected silkworm were transfected with a non-transposon vector pIZT/V5-His vector in vivo, and the blood, in which the hemocyte with green fluorescence could be observed, was added to the cultured BmN cells. Furthermore, normal BmN cells were infected with germinated N. bombycis, and the infected cells were transfected with pIZT/V5-His. Continuous fluorescence observations exposed that there were N. bombycis with green fluorescence in some N. bombycis-infected cells, and the extracted genome from the purified N. bombycis spore was used as templates. PCR amplification was carried out with a pair of primers for specifically amplifying the green fluorescence protein (GFP) gene; a specific product representing the gfp gene could be amplified. Expression of the GFP protein through Western blotting also demonstrated that the gfp gene was perfectly inserted into the genome of N. bombysis. These results illustrated that exogenous gene can be integrated into N. bombycis genome by mediating with a non-transposon vector. Our research not only offers a strategy for research on gene function of N. bombycis but also provides an important reference for constructing genetically modified microsporidium utilized for biocontrol of pests.

Effects of BmCPV Infection on Silkworm Bombyx mori Intestinal Bacteria.

The gut microbiota has a crucial role in the growth, development and environmental adaptation in the host insect. The objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus (BmCPV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female (male) larvae and BmCPV-infected female (male) larvae, respectively. In general, the microbial communities in the gut content of healthy larvae were dominated by Enterococcus, Delftia, Pelomonas, Ralstonia and Staphylococcus, however the abundance change of each genus was depended on the developmental stage and gender. Microbial diversity reached minimum at 144 h of fifth instar larvae. The abundance of Enterococcus in the females was substantially lower and the abundance of Delftia, Aurantimonas and Staphylococcus was substantially higher compared to the males. Bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. These results provided insight into the relationship between the gut microbiota and development of the BmCPV-infected silkworm.

A dithienosilole-based fluorescent chemosensor for multiple logic operations at the molecular level.

A chemosensor consisting of two terpyridines covalently linked to a dithienosilole unit (1) has been synthesized, and its optical and metal sensing properties have been investigated. Due to the metal-organic coordination function, 1 can bind with many transition metal ions and display different fluorescence responses that cause it to function as a "turn-off" fluorescent chemosensor. A significant bathochromic shift in the fluorescence spectra is observed in the presence of Zn(2+). Meanwhile, the emission of 1 is weakened upon exposure to Ag(+) and Fe(2+) and completely quenched by Ni(2+), Co(2+), and Cu(2+). Based on the observed results, several logic gates, such as XNOR, INHIBIT, and IMPLICATION, have been achieved by controlling the chemical inputs.

The gene expression profile of resistant and susceptible Bombyx mori strains reveals cypovirus-associated variations in host gene transcript levels.

High-throughput paired-end RNA sequencing (RNA-Seq) was performed to investigate the gene expression profile of a susceptible Bombyx mori strain, Lan5, and a resistant B. mori strain, Ou17, which were both orally infected with B. mori cypovirus (BmCPV) in the midgut. There were 330 and 218 up-regulated genes, while there were 147 and 260 down-regulated genes in the Lan5 and Ou17 strains, respectively. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment for differentially expressed genes (DEGs) were carried out. Moreover, gene interaction network (STRING) analyses were performed to analyze the relationships among the shared DEGs. Some of these genes were related and formed a large network, in which the genes for B. mori cuticular protein RR-2 motif 123 (BmCPR123) and the gene for B. mori DNA replication licensing factor Mcm2-like (BmMCM2) were key genes among the common up-regulated DEGs, whereas the gene for B. mori heat shock protein 20.1 (Bmhsp20.1) was the central gene among the shared down-regulated DEGs between Lan5 vs Lan5-CPV and Ou17 vs Ou17-CPV. These findings established a comprehensive database of genes that are differentially expressed in response to BmCPV infection between silkworm strains that differed in resistance to BmCPV and implied that these DEGs might be involved in B. mori immune responses against BmCPV infection.

Immune signaling pathways activated in response to different pathogenic micro-organisms in Bombyx mori.

The JAK/STAT, Toll, Imd, and RNAi pathways are the major signaling pathways associated with insect innate immunity. To explore the different immune signaling pathways triggered in response to pathogenic micro-organism infections in the silkworm, Bombyx mori, the expression levels of the signal transducer and activator of transcription (BmSTAT), spatzle-1 (Bmspz-1), peptidoglycan-recognition protein LB (BmPGRP-LB), peptidoglycan-recognition protein LE (BmPGRP-LE), argonaute 2 (Bmago2), and dicer-2 (Bmdcr2) genes after challenge with Escherichia coli (E. coli), Serratiamarcescens (Sm), Bacillus bombyseptieus (Bab), Beauveriabassiana (Beb), nucleopolyhedrovirus (BmNPV), cypovirus (BmCPV), bidensovirus (BmBDV), or Nosemabombycis (Nb) were determined using real-time PCR. We found that the JAK/STAT pathway could be activated by challenge with BmNPV and BmBDV, the Toll pathway could be most robustly induced by challenge with Beb, the Imd pathway was mainly activated in response to infection by E. coli and Sm, and the RNAi pathway was not activated by viral infection, but could be triggered by some bacterial infections. These findings yield insights into the immune signaling pathways activated in response to different pathogenic micro-organisms in the silkworm.