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Abstract: To explore the influence of the direct repeat sequence (DRS) in Bacillus chitinase genes on heterogonous expression in Escherichia coli, we cloned and sequenced the entire open reading frame (ORF) and upstream sequences of the chitinase B (chiB) and chitinase MY75 (chiMY75) from Bacillus thuringiensis and Bacillus licheniformis. A pair of 8-bp DRS was found upstream of each chi gene. Chi ORFs with a series of truncated DRS were cloned and transformed into E. coli XL-Blue. The activity of the transformants without the DRS were significantly higher in chitinase assays than transformants containing the DRS. SDS-PAGE showed that part and full deletion of the DRS increased chi gene expression by approximately 1.7 and 3.8-fold, respectively. Northern blotting revealed deletion of the DRS regions increased chiB and chiMY75 mRNA expression. Specific binding of DNA-binding factors in the E. coli cell lyaste was observed to both the chiB and chiMY75 promoter regions and DRS elements. This is the first investigation to demonstrate that heterologous expression of Bacillus chi genes in E. coli is negatively regulated by their upstream DRS regions, which act as cis-acting elements. [Copyright &y& Elsevier]

Abstract: Progranulin (PGRN) is abundantly expressed in epithelial cells, immune cells, neurons, and chondrocytes, and reportedly contributes to tumorigenesis. PGRN is a crucial mediator of wound healing and tissue repair. PGRN also functions as a neurotrophic factor and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. PGRN has been found to be a novel chondrogenic growth factor and to play an important role in cartilage development and inflammatory arthritis. Although research has shown that PGRN exhibits anti-inflammatory properties, the details about the exact molecular pathway of such effects, and, in particular, the PGRN binding receptor, have not been identified so far. Recently, researchers have shown that PGRN binds to tumor necrosis factor (TNF)-receptors (TNFR), interfering with the interaction between TNFα and TNFR. They further demonstrated that mice deficient in PGRN are susceptible to collagen-induced arthritis, an experimental model of rheumatoid arthritis, and that administration of PGRN reversed the arthritic process. An engineered protein made of three PGRN fragments (Atsttrin), displayed selective TNFR binding and was more active than natural PGRN. Both PGRN and Atsttrin prevented inflammation in various arthritis mouse models and inhibited TNFα-induced intracellular signaling pathways. Thus, PGRN is a key regulator of inflammation and it may mediate its anti-inflammatory effects, at least in part, by blocking TNF binding to its receptors. As we discuss here, TNFR-based interventions may both stimulate and suppress the growth of cancer cells, and the same may be true in analogy for Atsttrin as a new player. [Copyright &y& Elsevier]

Ershiwuwei Lvxue Pill (ELP, མགྲིན་མཚལ་ཉེར་ལྔ།), a traditional Tibetan medicine preparation, has been used hundreds of years for the clinical treatment of rheumatoid arthritis (RA) in the highland region of Tibet, China. However, the underlying mechanism of its therapeutic effect remains unclear. The present study aimed to investigate the potential pharmacological mechanisms of anti-arthritic effect of ELP. The main chemical constituents of ELP were analyzed by ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF-MS). Forty-eight male Wistar rats (220 ± 20 g) were randomly divided into six groups: normal group, collagen-induced arthritis (CIA) group, methotrexate group (1.05 mg/kg), ELP groups (115, 230 and 460 mg/kg). CIA rat models were assigned to evaluate the anti-RA activity of ELP by determining the paws swelling, arthritis score, organ coefficients of spleen and thymus, and histopathological analysis of knee joints of synovial tissues. The levels of TNF-α, IL-10, IL-6 and IL-17 in serum were measured by ELISA. In addition, mRNA and protein expression levels associated with JAK2/STAT3 signaling pathway in synovial tissues of CIA rats were detected by qRT-PCR, immunohistochemistry and Western blot analyses. Fourteen main chemical constituents of ELP were quantitatively determined by UPLC-Q-TOF-MS analysis. Treatment with ELP reduced the paw swelling, arthritis score and organ coefficients of spleen and thymus. Histopathological examination revealed the protective effects of ELP on CIA rats with knee joint injury. The levels of serum pro-inflammatory cytokines (TNF-α, IL-6 and IL-17) were markedly reduced while the anti-inflammatory cytokine IL-10 was significantly increased with the treatment of ELP. Further investigations showed ELP down-regulated the mRNA and protein expression levels of Bcl-2, whereas up-regulated Bax, SOCS1 and SOCS3. Meanwhile, the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 proteins from synovial tissues were dramatically decreased with the treatment of ELP, whereas no changes of the mRNA and protein expression levels of JAK2 and STAT3 were observed. These results indicated that ELP reduced the severity of arthritis and joint swelling, suggesting an antirheumatic effect on CIA rats. The possible mechanism is related to inhibiting inflammatory response and inducing apoptosis in synovial tissues by regulating JAK2/STAT3 signaling pathway. However, further in vivo and in vitro investigations are still needed to clarify the underlying mechanism of ELP in treating RA. Image 1 [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic inflammatory disease due to immune dysregulation that cannot be cured. The skin conditions of psoriasis negatively impact patients' quality of life worldwide. Qing Dai (Indigo Naturalis), a traditional Chinese medicine (TCM) processed from Strobilanthes cusia is a clinical medicine used for psoriasis patient in Taiwan and the gene overexpression of interleukin (IL)-17A could be notably reduced in skin lesions after using Qing Dai ointment and its alkaloid ingredients. To develop a potential anti-psoriatic phytopharmaceutical, an alkaloid-rich fraction named INM-A was prepared from Qing Dai. The chemical profile and anti-psoriatic activity of INM-A were analyzed and evaluated to define its in vitro mechanism and in vivo efficacy for psoriasis therapy. Dowex® 50WX4 hydrogen form resin was used for column chromatography to prepare INM-A. To track alkaloids, INM-A was conducted with Dragendorff's, Mayer's, and Wagner's reagents. HPLC and UV–Vis spectrophotometer were applied to analyze the chemical profile and relative total alkaloid content in INM-A. A psoriatic mouse model induced by imiquimod (IMQ) was performed to verify in vivo efficacy of INM-A. IL-17A-dominated cellular oxygen consumption rate, oxidative stress, and cytokines in keratinocytes were measured to clarify in vitro mechanism of INM-A. An alkaloid-rich fraction, INM-A, consisted of seven active alkaloid compounds 1 – 7 was obtained from Qing Dai. INM-A improved the skin condition severities in IMQ-induced psoriatic mice and decreased IL-17A in not only psoriatic mice but also polarized Th17 cells. In addition, INM-A targeted IL-17A to inhibit inflammation and OXPHOS-driven oxidative stress in human keratinocytes. Accordingly, INM-A manufactured from Qing Dai may be a promising lead phytopharmaceutical for further IL-17A-related inflammatory disease studies such as psoriasis. [Display omitted] • An alkaloid-rich phytopharmaceutical (INM-A) was prepared from Qing Dai. • INM-A could alleviate the severities of dermatitis in IMQ-induced psoriatic mice. • INM-A could decrease IL-17A productions in psoriatic mice and polarized Th17 cells. • INM-A targeted IL-17A to inhibit inflammation (IL-6 and IL-8) in HaCaT cells. • INM-A targeted IL-17A to inhibit OXPHOS-driven oxidative stress in HaCaT cells. [ABSTRACT FROM AUTHOR]

DNA damage is a serious threat to all living organisms and may be induced by environmental stressors. Previous studies have revealed that the tardigrade (Ramazzotius varieornatus) DNA damage suppressor protein Dsup has protective effects in human cells and tobacco. However, whether Dsup provides radiation damage protection more widely in crops is unclear. To explore the effects of Dsup in other crops, stable Dsup overexpression lines through Agrobacterium -mediated transformation were generated and their agronomic traits were deeply investigated. In this study, the overexpression of Dsup not only enhanced the DNA damage resistance at the seeds and seedlings stages, they also exhibited grain size enlargement and starch granule structure and cell size alteration by the scanning electron microscopy observation. Notably, the RNA-seq revealed that the Dsup plants increased radiation-related and abiotic stress-related gene expression in comparison to wild types, suggesting that Dsup is capable to coordinate normal growth and abiotic stress resistance in rice. Immunoprecipitation enrichment with liquid chromatography-tandem mass spectrometry (IP-LC-MS) assays uncovered 21 proteins preferably interacting with Dsup in plants, suggesting that Dsup binds to transcription and translation related proteins to regulate the homeostasis between DNA protection and plant development. In conclusion, our data provide a detailed agronomic analysis of Dsup plants and potential mechanisms of Dsup function in crops. Our findings provide novel insights for the breeding of crop radiation resistance. [Display omitted] • Dsup expression enhances UV stress tolerance in rice. • Dsup expression alters grain size, grain weight, and starch structures. • Dsup upregulates DNA repair-related genes to mediate DNA repair. • Dsup binds to transcriptional and translational related proteins to coordinate plant abiotic stress response and plant growth. [ABSTRACT FROM AUTHOR]

Abstract: Chitinase A (ChiA) produced by Bacillus thuringiensis subsp. colmeri 15A3 (Bt. 15A3) was expressed in Escherichia coli XL-Blue. The ChiA was purified using Sephadex G-200 and its molecular mass was estimated to be 36kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detection of chitinase activity on SDS-PAGE after protein renaturation indicated that the molecular mass of the protein band expressing chitinase activity was approximately 72kDa. This suggests that the dimeric form of ChiA is the enzymatically active form when glycol chitin is used as a substrate. ChiA has optimal activity at 50°C and retains most of its activity between 20 and 60°C. The optimum pH for ChiA activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. The enzyme activity was significantly inhibited by Ag+ and Zn2+. ChiA significantly inhibited the spore germination of four species of fungi. The median inhibitory concentrations (IC50) of ChiA on the spore germination of Penicillium glaucum and Sclerotinia fuckelian were 11.27 and 10.57μg/ml, respectively. In surface contamination bioassays, the crude ChiA protein (12.6mU) reduced the LC50 (50% lethal concentration) of the crystal protein of Bt. 15A3 against the larvae of Spodoptera exigua and Helicoverpa armigera. [Copyright &y& Elsevier]