The experimental study of mir-99a-5p negative regulation of TLR8 receptor mediated-mediated innate immune response in rabbit knee cartilage injury.

BACKGROUND: Traumatic cartilage injury is an important cause of osteoarthritis (OA) and limb disability, and toll-like receptors (TLRs) mediated innate immune response has been confirmed to play a crucial role in cartilage injury. In the previous study, we found that the activation of TLR8 molecules in injured articular cartilage was more obvious than other TLRs by establishing an animal model of knee impact injury in rabbits, and the changes of TLR8 molecules could significantly affect the process of articular cartilage injury and repair. OBJECTIVE: To verify how mir-99a-5p regulates TLR8 receptor mediated innate immune response to treat traumatic cartilage injury. METHODS: The impact of a heavy object on the medial condyle of the rabbit's knee joint caused damage to the medial condylar cartilage. Through pathological and imaging analysis, it was demonstrated whether the establishment of an animal model of traumatic cartilage injury was successful. Establishing a cell model by virus transfection of chondrocytes to demonstrate the role of TLR8 in the innate immune response to impact cartilage injury. Through transcriptome sequencing, potential targets of TLR8, mir-99a-5p, were predicted, and basic experiments were conducted to demonstrate how they interact with innate immune responses to impact cartilage damage. RESULTS: TLR8 is a receptor protein of the immune system, which is widely expressed in immune cells. In our study, we found that TLR8 expression is localized in lysosomes and endosomes. Mir-99a-5p can negatively regulate TLR8 to activate PI3K-AKT molecular pathway and aggravate cartilage damage. Inhibiting TLR8 expression can effectively reduce the incidence of articular cartilage damage. CONCLUSION: Based on the results from this study, mir-99a-5p may be an effective molecular marker for predicting traumatic cartilage injury and targeting TLR8 is a novel and promising approach for the prevention or early treatment of cartilage damage.

Dopamine alters phage morphology to exert an anti-infection effect.

Antiviral drug development is important for human health, and the emergence of novel COVID-19 variants has seriously affected human lives and safety. A bacteriophage-a bacterial virus with a small and simple structure-is an ideal experimental candidate for studying the interactions between viruses and their hosts. In this study, the effects and mechanisms of catecholamines on phages were explored, and dopamine (DA) was found to have general and efficient anti-infection effects. A clear dose-dependent effect was observed when different phages were treated with DA, with higher DA concentrations exhibiting stronger anti-phage activity. The half maximal inhibitory concentration values of DA for vB-EcoS-IME167, T4 Phage, and VMY22 were determined as 0.26, 0.12, and 0.73 mg mL-1, respectively. The anti-phage effect of DA increased with treatment duration. In addition, the anti-infection activities of DA against vB-EcoS-IME167, T4 Phage, and VMY22 were increased by 105, 104, and 104 folds compared to that of the control. This ability of DA was observed only in phages and not in the host bacteria. Morphological changes of phages were observed under transmission electron microscopy following their treatment with DA, and considerable changes in adsorption were confirmed via quantitative reverse transcription polymerase chain reaction. These results suggest that the anti-phage effect of DA is primarily due to the destruction of the external structure of the phage. This study, to the best of our knowledge, is the first to report the universal anti-phage infection effect of dopamine, which provides novel information regarding DA and forms a basis for further research and development of antiviral drugs. Moreover, it provides a new perspective for the research about the defense and counter-defense of bacteria and bacteriophages.

Mechanisms underlying the inhibitory effects of Cd2+ on prodigiosin synthesis in Serratia marcescens KMR-3.

Prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), a red-colored microbial pigment, is produced in large quantities by Serratia marcescens KMR-3. This bacterium can grow in a medium with a Cd2+ concentration of 500 mg/L, but it does not produce prodigiosin when the Cd2+ concentration in the medium is higher than 140 mg/L. Therefore, we investigated the mechanisms by which Cd2+ inhibits prodigiosin synthesis. Upon addition of Cd2+ to the medium, the expression of the prodigiosin (pig) gene cluster was significantly downregulated. Simultaneously, genes encoding proteins related to the synthesis of arginine and proline(prodigiosin precursors) were significantly downregulated, while the degradation-related genes were upregulated. Furthermore, PigF, which encodes a key enzyme involved in the synthesis of 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde and PigC, which encodes a key enzyme involved in the last step of prodigiosin synthesis, were downregulated by 80% and 55%, respectively, following Cd2+ treatment. As PigC and PigF are located on the cell membrane and are involved in the final steps of prodigiosin synthesis, the cell membrane might be presumed to be the site of prodigiosin synthesis. The bacterial membrane exhibited different degrees of elongation, folding, fragmentation, and sagging after the addition of Cd2+, while likely destroying the site of prodigiosin synthesis.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo.

Intracellular Fe3+ amount is one of the critical determinants of human health. The development of simple and effective probes for the quantitative detection of Fe3+in vivo is of great significance for the early diagnosis of disease or disorder associated with iron deficiency or overload. In this study, remarkable carbon dots, which can serve as a biosensor for efficient intracellular Fe3+ detection, were synthesized by hydrothermal carbonization of Fusobacterium nucleatum, an anaerobic bacterium. The achieved F. nucleatum-carbon dots (Fn-CDs) possessed the features of strong fluorescence, high stability and excellent biocompatibility. The obtained Fn-CDs could easily internalize into both plant cells and human cells with excellent ability for cell tracking and biomedical labeling. The fluorescence of Fn-CDs could still remain for another 24 hours after penetrating into cells. Furthermore, the fluorescent Fn-CDs were very sensitive to the presence of Fe3+ ions even in cells, exhibiting great promising applications in in vivo detection of Fe3+ ions. In addition, the Fn-CDs posed no harm to the mice, being circulated and excreted within a short time, making the Fn-CDs an excellent candidate for bioimaging and biosensing in vivo.

The strong inhibitory effect of combining anti-cancer drugs AT406 and rocaglamide with blue LED irradiation on colorectal cancer cells.

There is still no satisfying method to treat colorectal cancer (CRC) currently. Inspired by cocktail therapy, the combination of 465 nm blue LED irradiation and two multi-target anticancer agents AT406 and Rocaglamide has been investigated as an innovative way to treat colorectal cancer cells in vitro. It showed a strong inhibitory effect on colorectal cancer cells, and its side effects on human normal cells are negligible. When applied to HCT116 cells, it can achieve an apoptotic rate up to 95%. It is also seen to significantly inhibit proliferation of HT29 cells. Furthermore, little to no cell inhibition or damage of normal MRC-5 cells were seen after treatment. The combination of blue LED irradiation and two anti-cancer drugs causes apoptosis of colorectal cancer cells by activating the apoptotic pathway, inhibiting autophagy and proliferation pathways as well as the production of reactive oxygen species (ROS).

Screening GLP-1 Receptor Ligands from Natural Products in Herbs through High-Content Technique.

AIM AND OBJECTIVE: Screening of active components from a natural product, especially from a crude extract, is a great challenge. To avoid potential activity interference of the N-terminus modification in the most common constructs based on GCPRs labeled with GFP technology, a Cterminus tGFP-labeled hGLP-1 receptor containing recombinant cell line hGLP-1R-tGFP was constructed and tried to be used in the screening of natural products from Chinese herb. MATERIALS AND METHODS: The GLP1 receptor gene was amplified and the inserts pCMV6-AC-tGFP and tGFP were fused at the C-terminus of GLP1 receptor to construct a recombinant plasmid. The recombinant was transfected into U2OS cell and selected with antibiotics and flow cytometry. The constructed cell line was named as hGLP-1R-tGFP cell line. The expression levels of GLP-1R-tGFP protein were confirmed by western-blot. The fluorescence imaging of re-distribution from diffusing to aggregate spots inside the cells was quantitated and analyzed by High Content Screening (HCS) assay. Meanwhile, the specificity, stability and C-terminus function of hGLP-1R-tGFP cell line were characterized. In order to allow the recombinant cell line of hGLP-1R-tGFP to be suitable in highcontent system of Arrayscan-infinity-700 in screening mode, several conditions have also been optimized. In the end, a total of 100 crude extract samples provided by the Yunnan Institute of Materia Medica have been screened with this method. RESULTS: Upon the activation of GLP-1 receptors by Exendin 4, fluorescent patches appeared on the cell membrane and subsequently internalized to form fluorescent aggregates inside the cells under fluorescent microscopy examination. The agonistic activity, sensitivity and specificity of the formation of fluorescent aggregate spot in hGLP-1R-tGFP cells have been confirmed by the activation of GLP-1R using the GLP-1analogues. The agonistic effects of GLP-1 analogues are blocked by a GLP-1R antagonist, Exendin9-39. The downstream of GLP-1 pathway, the activation of adenylate cyclase and the raising of cellular cAMP levels, remained intact in these tGFP modified C-terminus GLP-1 receptor cells. Meanwhile, a total of 100 crude extract samples from Chinese herbs have been screened by this method to find new active ingredients. CONCLUSION: Combined with High Content Screening image and data automatic acquisition processing, a new screening assay based on a recombinant U2OS cell line which GFP labeled at the C terminus of GLP1 receptor has been developed. GLP-1R agonist activity in extracts of Astragalus propinquus and Panax notoginseng from Chinese herbs has been determined by this method.

Bacteria-derived fluorescent carbon dots for highly selective detection of p-nitrophenol and bioimaging.

p-Nitrophenol (p-NP) pollutants are widely present in soil and aquatic environments and can seriously impair the health of living beings. Hence, a rapid, sensitive, and selective method for p-NP detection is urgently needed. Herein, for the first time, we successfully synthesized fluorescent carbon dots (CDs) from Bacillus cereus (BC) via a one-step hydrothermal process. The obtained CDs-BC can be applied as a rapid, highly selective, and sensitive sensor for p-NP detection. The fluorescence quenching efficiency of the CD-BC sensor exhibited excellent linear responses with p-NP concentrations at both 0.3-6.5 µM and 6.5-30 µM, with a detection limit of 0.11 µM. The mechanism of p-NP detection is based on the inner filter effect (IFE). Preliminary bacteria, cell, and animal studies showed that the as-prepared CDs-BC possess high photostability, excellent biocompatibility, low or no biotoxicity, and multicolor fluorescence emission properties; furthermore, they can be rapidly excreted from the body of mice, which suggests their potential for applications in the biomedical field.

Evaluation of the significance of hepatic functional reserve for the operation of liver cancer complicated with cirrhosis.

PURPOSE: To evaluate the significance of hepatic functional reserve for the operation of liver cancer complicated with cirrhosis. METHODS: Fifty-six patients suffering from liver cancer complicated with cirrhosis were divided into three levels, A, B and C, according to Child-Pugh grading system. Based on indocyanine green retention rate at 15 min (ICGR15) value, patients were divided into three intervals, ≤15%, 15-25% and ≥25%. According to the existence of complications, patients were divided into the complication group and the no complication group. RESULTS: Child-Pugh grading included 50 cases of level A, 45 cases of B and 29 cases of C. ICGR15 value included 47 cases of ≤15% interval, 47 cases of 15-25% and 30 cases of ≥25%. As the IGCR15 value increased, the levels of all indicators were obviously increased. Among the 124 patients, 35 cases (28.23%) suffered complications The median follow-up time was 25.0 months. The survival rate of the complication group was 60.00% (21 cases), significantly lower than that of the no complication group (84.27%). Child-Pugh grading of the complication group included 4 cases of level A, 12 cases of B and 19 cases of C. ICGR15 value included 15 cases of 15~25% interval and 20 cases of ≥25%. CONCLUSION: Child-Pugh grading and ICGR15 value can both reflect hepatic functional reserve and are of great clinical significance for complication and survival. There is a fairly good relevance between ICGR15 and levels of AFP, ALT and indicators of liver fibrosis.

Isolation and characterization of wetland VSW-3, a novel lytic cold-active bacteriophage of Pseudomonas fluorescens.

Wetlands are often called the "kidneys of the Earth" and contribute substantially to environmental improvement. Pseudomonas fluorescens is a major contaminant of milk products and causes the spoilage of refrigerated foods and fresh poultry. In this study, we isolated and characterized a lytic cold-active bacteriophage named VSW-3 together with P. fluorescens SW-3 cells from the Napahai wetland in China. Electron microscopy showed that VSW-3 had an icosahedral head (56 nm) and a tapering tail (20 nm × 12 nm) and a genome size of approximate 40 kb. On the basis of the top-scoring hits in the BLASTP analysis, VSW-3 showed a high degree of module similarity to the Pseudomonas phages Andromeda and Bf7. The latent and burst periods were 45 and 20 min, respectively, with an average burst size of 90 phage particles per infected cell. The pH and thermal stability of VSW-3 were also explored. The optimal pH was found to be 7.0 and the activity decreased rapidly when the temperature exceeded 60 °C. VSW-3 is a cold-active bacteriophage, hence, it is important to research its ability to prevent product contamination caused by P. fluorescens and to characterize its relationship with its host P. fluorescens in the future.

Complete genome sequence of the cold-active bacteriophage VMY22 from Bacillus cereus.

The cold-active bacteriophage VMY22, belonging to the Podoviridae family, was isolated from Mingyong Glacier in China. Sequence analysis revealed that the genome is 18,609 bp long, with an overall G + C content of 36.4 mol%, and 25 open reading frames (ORFs). The sequence contains 46 potential promoters, 6 transcription terminators, and no tRNAs. Most of the ORFs show a high degree of similarity to B103 (NC_004165). Two noteworthy findings were made. First, one of the predicted proteins, ORF 19, shows high sequence similarity to the bacteriocin biosynthesis protein from Bacillus cereus. From this information, we propose that the VMY22 phage is at an intermediate phase in its coevolution with its bacterial host. Second, seven of the hypothetical proteins appear to be unique to this cold-active B. cereus phage (i.e., not found in temperate-active B. cereus phages). These observations add to our current knowledge about the coevolution of bacteriophages and their hosts. The identification of a novel group of gene and protein structures and functions will lead to a better understanding of cold-adaptation mechanisms in bacteria and their bacteriophages.

Localized primary gastrointestinal diffuse large B cell lymphoma received a surgical approach: an analysis of prognostic factors and comparison of staging systems in 101 patients from a single institution.

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) represents the most common histological subtype of primary gastrointestinal lymphoma and is a heterogeneous group of disease. Prognostic characterization of individual patients is an essential prerequisite for a proper risk-based therapeutic choice. METHODS: Clinical and pathological prognostic factors were identified, and predictive value of four previously described prognostic systems were assessed in 101 primary gastrointestinal DLBCL (PG-DLBCL) patients with localized disease, including Ann Arbor staging with Musshoff modification, International Prognostic Index (IPI), Lugano classification, and Paris staging system. RESULTS: Univariate factors correlated with inferior survival time were clinical parameters [age>60 years old, multiple extranodal/gastrointestinal involvement, elevated serum lactate dehydrogenase and ß2-microglobulin, and decreased serum albumin], as well as pathological parameters (invasion depth beyond serosa, involvement of regional lymph node or adjacent tissue, Ki-67 index, and Bcl-2 expression). Major independent variables of adverse outcome indicated by multivariate analysis were multiple gastrointestinal involvement. In patients unfit for Rituximab but received surgery, radical surgery significantly prolonged the survival time, comparing with alleviative surgery. Addition of Rituximab could overcome the negative prognostic effect of alleviative surgery. Among the four prognostic systems, IPI and Lugano classification clearly separated patients into different risk groups. IPI was able to further stratify the early-stage patients of Lugano classification into groups with distinct prognosis. CONCLUSIONS: Radical surgery might be proposed for the patients unfit for Rituximab treatment, and a combination of clinical and pathological staging systems was more helpful to predict the disease outcome of PG-DLBCL patients.

[Clinical characteristics and prognostic analysis of 90 patients with primary gastro-intestinal marginal zone lymphoma].

OBJECTIVE: To evaluate the clinical characteristics and prognostic factors of patients with primary gastro-intestinal marginal zone lymphoma (MALT). METHODS: Retrospective analysis was performed in 90 patients diagnosed with primary gastro-intestinal MALT lymphoma clinical characteristics and survival analyses. RESULTS: Among 90 patients, 78 cases were originated from the stomach and 12 cases with extra-gastric origin. Eighty patients were classified as low-risk (IPI score 0-2), and 10 patients high-risk (IPI score 3-5). Compared to gastric MALT patients, extra-gastric cases presented with higher IPI score (7.7% vs 33.3%, P=0.025) and higher Hp infection rate (50.0% vs 87.2%, P<0.01). Treatment options for low risk patients (IPI score 0-2) included Hp eradication, surgery, radiotherapy and chemotherapy. Chemotherapy could improve progression-free survival (PFS) in low-risk patients. For high-risk patients, those receiving chemotherapy had 100% 3-year overall survival (OS). Univariate analysis revealed that ECOG (P=0.006), Mussh-off staging (P=0.008), IPI score (P=0.000), elevated LDH (P=0.019) and chemotherapy (P=0.026) were correlated with PFS. Multivariate analysis showed that higher IPI score (IPI 3-5) (OR=8.325, 95% CI 3.171-21.853, P=0.000) and chemotherapy (OR=0.319, 95% CI 0.121-0.838, P=0.020) were independent prognostic factors for PFS. ECOG (≥ 2) was independent prognostic factor for OS (OR=5.092, 95%CI 1.005-25.788, P=0.049). CONCLUSION: Primary gastro-intestinal MALT lymphoma was an indolent subtype of non-Hodgkin's lymphoma. Patients usually had low risk IPI and achieved long-term survival. Frontline therapy for low-risk patients was radiotherapy or Hp eradication, and chemotherapy for high-risk ones.

Codon 104 variation of p53 gene provides adaptive apoptotic responses to extreme environments in mammals of the Tibet plateau.

Mutational changes in p53 correlate well with tumorigenesis. Remarkably, however, relatively little is known about the role that p53 variations may play in environmental adaptation. Here we report that codon asparagine-104 (104N) and glutamic acid-104 (104E), respectively, of the p53 gene in the wild zokor (Myospalax baileyi) and root vole (Microtus oeconomus) are adaptively variable, meeting the environmental stresses of the Tibetan plateau. They differ from serine-104 (104S) seen in other rodents, including the lowland subterranean zokor Myospalax cansus, and from serine 106 (106S) in humans. Based on site-directed mutational analysis in human cell lines, the codon 104N variation in M. baileyi is responsible for the adaptive balance of the transactivation of apoptotic genes under hypoxia, cold, and acidic stresses. The 104E p53 variant in Microtus oeconomus suppresses apoptotic gene transactivation and cell apoptosis. Neither 104N nor 104E affects the cell-cycle genes. We propose that these variations in p53 codon 104 are an outcome of environmental adaptation and evolutionary selection that enhance cellular strategies for surviving the environmental stresses of hypoxia and cold (in M. baileyi and M. oeconomus) and hypercapnia (in M. baileyi) in the stressful environments of the Qinghai-Tibet plateau.

Distinct post-transcriptional regulation of Igfbp1 gene by hypoxia in lowland mouse and Qinghai-Tibet plateau root vole Microtus oeconomus.

Our previous study revealed the particular expression patterns of insulin-like growth factor 1 (IGF1) and insulin-like growth factor binding protein 1 (IGFBP1) in the Qinghai-Tibet plateau root vole (Microtus oeconomus) under hypoxic challenge. Here we report the molecular mechanisms of Igf gene regulation associated with adaptation to hypoxia. M. oeconomus IGF1 and IGFBP1 were shown to be highly conserved. Hypoxia (8.0% O2, 6h) did not change the liver-derived Igf1 expression in either M. oeconomus or mouse. Hypoxia significantly upregulated hepatic Igfbp1 gene expression and IGFBP1 levels in the liver and plasma of the mouse, but not in M. oeconomus. A functional U-rich element in the 3' untranslated region was found in mouse Igfbp1 mRNA, which was associated with Igfbp1 mRNA stabilization and upregulation under hypoxia, and this U-rich element was eliminated in the M. oeconomus Igfbp1, resulting in blunted Igfbp1 mRNA upregulation, which might be understood as a sequence variation modified during molecular evolution under hypoxia.

Discovery of novel quinolinone adenosine A2B antagonists.

A novel series of quinolinone-based adenosine A(2B) receptor antagonists was identified via high throughput screening of an encoded combinatorial compound collection. Synthesis and assay of a series of analogs highlighted essential structural features of the initial hit. Optimization resulted in an A(2B) antagonist (2i) which exhibited potent activity in a cAMP accumulation assay (5.1 nM) and an IL-8 release assay (0.4 nM).