Pueraria lobata-derived exosome-like nanovesicles alleviate osteoporosis by enhacning autophagy.

Osteoporosis (OP) is the most common bone disorder worldwide, especially in postmenopausal women. However, many OP drugs are not suitable for long term use due to major adverse effects. Therefore, there is an urgent need to identify more effective and safe therapeutic drugs. Pueraria lobata has been reported to promote osteoblast growth in bone regeneration, but the exact mechanisms still need further exploration. The current study found that Pueraria lobata-derived exosome-like nanovesicles (PELNs) promoting primary human bone mesenchymal stem cells (hBMSCs) differentiation and mineralization both in vitro and in ovariectomized (OVX)-induced osteoporotic rats. Interestingly, the relative abundance of harmful strains significantly decreased in the intestine of the osteoporosis SD rat model administrated PELNs via the regulation of trimethylamine-N-oxide (TMAO), a metabolite of gut microbiota. Moreover, RNA sequencing revealed that the osteogenic activity of PELNs is revealed to autophagy signaling. In vitro and in vivo experiments also showed that the treatment with PELNs promoted the differentiation and function of hBMSCs by elevating autophagy via the degradation of TMAO. Collectively, PELNs demonstrate promise as a therapeutic approach for OP, with TMAO emerging as a potential target of OP treatment.

Toxoplasma gondii excretory/secretory proteins promotes osteogenic differentiation of bone marrow mesenchymal stem cells via aerobic glycolysis mediated by Wnt/ß­catenin signaling pathway.

Toxoplasma gondii excretory/secretory proteins (TgESPs) are a group of proteins secreted by the parasite and have an important role in the interaction between the host and Toxoplasma gondii (T. gondii). They can participate in various biological processes in different cells and regulate cellular energy metabolism. However, the effect of TgESPs on energy metabolism and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) has remained elusive. In the present study, TgESPs were extracted from the T. gondii RH strain and used to treat BMSCs to observe the effect of TgESPs on energy metabolism and osteogenic differentiation of BMSCs and to explore the molecular mechanisms involved. The osteogenic differentiation and energy metabolism of BMSCs were evaluated using Alizarin Red S staining, qRT-PCR, western blot, immunofluorescence and Seahorse extracellular flux assays. The results indicated that TgESPs activated the Wnt/ß­catenin signaling pathway to enhance glycolysis and lactate production in BMSCs, and promoted cell mineralization and expression of osteogenic markers. In conclusion, the present study uncovered the potential mechanism by which TgESPs regulate BMSCs, which will provide a theoretical reference for the study of the function of TgESPs in the future.

Exosome-like Nanovesicles Derived from the Mucilage of Pinctada Martensii Exhibit Antitumor Activity against 143B Osteosarcoma Cells.

Osteosarcoma is prone to metastasis and has a low long-term survival rate. The drug treatment of osteosarcoma, side effects of treatment drugs, and prognosis of patients with lung metastasis continue to present significant challenges, and the efficacy of drugs used in the treatment of osteosarcoma remains low. The development of new therapeutic drugs is urgently needed. In this study, we successfully isolated Pinctada martensii mucilage exosome-like nanovesicles (PMMENs). Our findings demonstrated that PMMENs inhibited the viability and proliferation of 143B cells, induced apoptosis, and inhibited cell proliferation by suppressing the activation of the ERK1/2 and Wnt signaling pathways. Furthermore, PMMENs inhibited cell migration and invasion by downregulating N-cadherin, vimentin, and matrix metalloprotease-2 protein expression levels. Transcriptomic and metabolomic analyses revealed that differential genes were co-enriched with differential metabolites in cancer signaling pathways. These results suggest that PMMENs may exert anti-tumor activity by targeting the ERK1/2 and Wnt signaling pathways. Moreover, tumor xenograft model experiments showed that PMMENs can inhibit the growth of osteosarcoma in mice. Thus, PMMENs may be a potential anti-osteosarcoma drug.

Endothelial progenitor cell derived exosomes mediated miR-182-5p delivery accelerate diabetic wound healing via down-regulating PPARG.

Diabetic wound is one of the most common and serious complications of diabetes, which is characterized by abnormal number and quality of wound repair related cells. Previous studies have shown that human endothelial progenitor cells derived exosomes (EPCs-EXO) can promote diabetic wound healing through modulating vascular endothelial cell function. The purpose of this study was to investigate the biological effects and molecular mechanisms of EPCs-EXO on diabetic wound healing. The regulation of EPCs-EXO on human immortalized epidermal cell line HaCaT in high glucose (HG) environment was evaluated. Our data showed that EPCs-EXO promoted the proliferation, migration, while inhibited apoptosis of HaCaTs challenged by HG via elevating miR-182-5p expression level in vitro. Skin wound healing was significantly enhanced by EPCs-EXO in diabetic mice. Moreover, bioinformatics analyses and luciferase reporter assay indicated that exosomal miR-182-5p was bound to PPARG 3' UTR sequence and inhibited the expression of PPARG. Collectively, our findings provided a new role of EPCs-EXO in the clinical treatment of diabetic skin wounds. Diabetic wound is one of the most common and serious complications of diabetes, which is characterized by abnormal number and quality of wound repair related cells. Previous studies have shown that human endothelial progenitor cells derived exosomes (EPCs-EXO) can promote diabetic wound healing through modulating vascular endothelial cell function. The purpose of this study was to investigate the biological effects and molecular mechanisms of EPCs-EXO on diabetic wound healing. The regulation of EPCs-EXO on human immortalized epidermal cell line HaCaT in high glucose (HG) environment was evaluated. Our data showed that EPCs-EXO promoted the proliferation, migration, while inhibited apoptosis of HaCaTs challenged by HG via elevating miR-182-5p expression level in vitro. Skin wound healing was significantly enhanced by EPCs-EXO in diabetic mice. Moreover, bioinformatics analyses and luciferase reporter assay indicated that exosomal miR-182-5p was bound to PPARG 3' UTR sequence and inhibited the expression of PPARG. Collectively, our findings provided a new role of EPCs-EXO in the clinical treatment of diabetic skin wounds.

DPA promotes hBMSCs osteogenic differentiation by miR-9-5p/ERK/ALP signaling pathway.

Docosahexaenoic acid (DHA) has been reported potentiate osteogenic differentiation, while Docosapentaenoic acid (DPA), another Omega-3 fatty acid, its contribution to the osteogenic differentiation of human bone-marrow-derived mesenchymal stromal cells (hBMSCs) is not entirely elucidated. The Alizarin Red S (ARS) staining and the expression of osteogenesis­associated genes were analyzed during osteogenic induction by DPA. Then, bioinformatics analysis and dual luciferase reporter assays were investigated to confirm the interactions between miR-9-5p and alkaline phosphatase (ALP). miR-9-5p mimics / inhibitor were transfected to human hBMSCs and the osteogenic assay above was also performed. Furthermore, DPA significantly promoted the phosphorylation of ERK via miR-9-5p. PD98059, a highly specific and potent ERK1/2 inhibitor, inhibited the activation of ALP and partially reversed the role of DPA during osteogenic differentiation. These data indicated that DPA promoted osteogenic differentiation of hBMSCs potentially through miR-9-5p/ERK/ALP signaling pathway, providing a potentially useful therapeutic strategy for patients to improve bone loss.

Toxoplasma gondii Induces Apoptosis via Endoplasmic Reticulum Stress-Derived Mitochondrial Pathway in Human Small Intestinal Epithelial Cell-Line.

Toxoplasma gondii, an intracellular protozoan parasite that infects one-third of the world's population, has been reported to hijack host cell apoptotic machinery and promote either an anti- or proapoptotic program depending on the parasite virulence and load and the host cell type. However, little is known about the regulation of human FHs 74 small intestinal epithelial cell viability in response to T. gondii infection. Here we show that T. gondii RH strain tachyzoite infection or ESP treatment of FHs 74 Int cells induced apoptosis, mitochondrial dysfunction and ER stress in host cells. Pretreatment with 4-PBA inhibited the expression or activation of key molecules involved in ER stress. In addition, both T. gondii and ESP challenge-induced mitochondrial dysfunction and cell death were dramatically suppressed in 4-PBA pretreated cells. Our study indicates that T. gondii infection induced ER stress in FHs 74 Int cells, which induced mitochondrial dysfunction followed by apoptosis. This may constitute a potential molecular mechanism responsible for the foodborne parasitic disease caused by T. gondii.

[Clinical analysis of 23 patients with coronavirus disease 2019 in Xinyang City of Henan Province].

OBJECTIVE: To analyze the epidemiological characteristics and clinical features of the patients with coronavirus disease 2019 (COVID-19), so as to provide basis for clinical diagnosis. METHODS: The epidemiology, clinical symptoms, laboratory and radiologic data of 23 patients with COVID-19 admitted to the Fifth People's Hospital of Xinyang City from January 22nd to January 29th, 2020 were retrospectively analyzed. RESULTS: There was 23 patients with COVID-19, with 15 men and 8 women, and the median age was 46.0 (40.5, 52.0) years old (ranged from 27 years old to 80 years old). Nine patients had basic disease (39.1%), including hypertension (17.4%), cardiovascular diseases (17.4%), diabetes (8.7%), hypothyroidism (4.3%) and past history of tuberculosis (4.3%). All the 23 patients had contact history in Wuhan area or with confirmed cases. Clinical symptoms included fever (100%), cough (69.6%), expectoration (43.5%), myalgia (26.1%), headache (17.4%) and dyspnea (17.4%), and the less common symptom was diarrhea (4.3%). Blood routine tests showed leukocytopenia in 11 patients (47.8%), normal leukocyte counts in 10 patients (43.5%), and leukocytosis in 2 patients (8.7%); lymphopenia was found in 13 patients (56.5%). All 23 patients had different degrees of infective lesions in chest CT, with 7 patients (30.4%) on one side and 16 patients (69.6%) on both sides. There were 19 mild patients, 4 severe patients, and no critical or death case. Complications included acute respiratory distress syndrome (17.4%). No patient was reported with liver, kidney or heart dysfunction or secondary infection. CONCLUSIONS: Epidemic history of contact, fever, pneumonia signs of chest CT, normal or decreased count of leukocyte and lymphopenia are the clinical basis for diagnosis of COVID-19. However, at present, the treatment of patients has not been completed, and the effective treatment strategy and final prognosis are unclear.

Investigation and optimization of particle dimensions for needle trap device as an exhaustive active sampler.

Various needle trap devices (NTDs) with different designs have been developed during the past decade. A theoretical model on the fundamentals of the NTD was recently proposed, which employed the theory of frontal (gas-solid) chromatography to describe the sampling process. In the current work, different types of sorbent particles with different dimensions were packed into the needle as the adsorbent. The effects of particle dimensions, which would affect the packing density and consequently affect the capacity, the extraction efficiency and desorption efficiency of the NTD were experimentally investigated and the proposed theory was validated. The results demonstrated that NTDs packed with small particles possess higher extraction capacity and efficiency but much higher resistances to flow as well. The higher resistance did not necessarily result in poor desorption efficiency. The observed relationships among those physical parameters provide valuable guidance on how to design a NTD with high performance for future applications.

Development of a unique small molecule modulator of CXCR4.

BACKGROUND: Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. METHODOLOGY/PRINCIPAL FINDINGS: We describe the actions of N,N'-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using (18)F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. CONCLUSIONS/SIGNIFICANCE: We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.

C6-C8 bridged epothilones: consequences of installing a conformational lock at the edge of the macrocycle.

A series of conformationally restrained epothilone analogues with a short bridge between the methyl groups at C6 and C8 was designed to mimic the binding pose assigned to our recently reported EpoA-microtubule binding model. A versatile synthetic route to these bridged epothilone analogues has been successfully devised and implemented. Biological evaluation of the compounds against A2780 human ovarian cancer and PC3 prostate cancer cell lines suggested that the introduction of a bridge between C6-C8 reduced potency by 25-1000 fold in comparison with natural epothilone D. Tubulin assembly measurements indicate these bridged epothilone analogues to be mildly active, but without significant microtubule stabilization capacity. Molecular mechanics and DFT energy evaluations suggest the mild activity of the bridged epo-analogues may be due to internal conformational strain.

Bioinspired synthesis of hirsutellones A, B, and C.

The total synthesis of hirsutellones A (1), B (2), and C (3) has been achieved through a bioinspired late-stage sequence starting from advanced intermediate 6. The sequence proceeded via labile intermediate 17,1'-dehydrohirsutellone B (5) and delivered, in addition to the natural products (1-3), hirsutellone analogue 1',2',17-epi-hirsutellone C (1',2',17-epi-3).

Dipyrimidine amines: a novel class of chemokine receptor type 4 antagonists with high specificity.

The C-X-C chemokine receptor type 4 (CXCR4)/stromal cell derived factor-1 (SDF-1 or CXCL12) interaction and the resulting cell signaling cascade play a key role in metastasis and inflammation. On the basis of the previously published CXCR4 antagonist 5 (WZ811), a series of novel nonpeptidic anti-CXCR4 small molecules have been designed and synthesized to improve potency. Following a structure-activity profile around 5, more advanced compounds in the N,N'-(1, 4-phenylenebis(methylene)) dipyrimidin-2-amines series were discovered and shown to possess higher CXCR4 binding potential and specificity than 5. Compound 26 (508MCl) is the lead compound and exhibits subnanomolar potency in three in vitro assays including competitive binding, Matrigel invasion and Gα(i) cyclic adenosine monophosphate (cAMP) modulation signaling. Furthermore, compound 26 displays promising effects by interfering with CXCR4 function in three mouse models: paw inflammation, Matrigel plug angiogenesis, and uveal melanoma micrometastasis. These data demonstrate that dipyrimidine amines are unique CXCR4 antagonists with high potency and specificity.

Fundamentals and applications of needle trap devices: a critical review.

The needle trap device (NTD) is an extraction trap that contains a sorbent inside a small needle, through which fluid can be actively drawn into and out of by a gas-tight syringe or pump, or analytes can be introduced passively to the trap by diffusion. The needle trap (NT) is a potentially solventless sampling technique/sample preparation and introduction device. Both fluid-borne analytes and particles can be trapped inside the needle and then adsorbed analytes are desorbed in an inlet of analytical instrument and introduced for identification and quantification. The fluid may be either gaseous or liquid. The objectives of this critical review are to summarize the theory of the sampling process for both active and passive time-average extraction modes in addition to outlining the evolution of the technology and main applications.

Design and synthesis of C6-C8 bridged epothilone A.

A conformationally restrained epothilone A analogue (3) with a short bridge between methyl groups at C6 and C8 was designed and synthesized. Preliminary biological evaluation indicates 3 to be only weakly active (IC50 = 8.5 microM) against the A2780 human ovarian cancer cell line.

Discovery of small molecule CXCR4 antagonists.

In light of a proposed molecular mechanism for the C-X-C chemokine receptor type 4 (CXCR4) antagonist 1 (AMD3100), a template with the general structure 2 was designed, and 15 was identified as a lead by means of an affinity binding assay against the ligand-mimicking CXCR4 antagonist 3 (TN14003). Following a structure-activity profile around 15, the design and synthesis of a series of novel small molecular CXCR4 antagonists led to the discovery of 32 (WZ811). The compound shows subnanomolar potency (EC50 = 0.3 nM) in an affinity binding assay. In addition, when subjected to in vitro functional evaluation, 32 efficiently inhibits CXCR4/stromal cell-derived factor-1 (SDF-1)-mediated modulation of cyclic adenosine monophophate (cAMP) levels (EC50 = 1.2 nM) and SDF-1 induced Matrigel invasion (EC50 = 5.2 nM). Molecular field topology analysis (MFTA), a 2D quantitative structure-activity relationship (QSAR) approach based on local molecular properties (Van der Waals radii (VdW), atomic charges, and local lipophilicity), applied to the 32 series suggests structural modifications to improve potency.

Nonpeptide inhibitors of measles virus entry.

Measles virus (MV) is one of the most infectious pathogens known. Despite the existence of a vaccine, over 500,000 deaths/year result from MV or associated complications. Anti-measles compounds could conceivably reverse these statistics. Previously, we described a homology model of the MV fusion protein trimer and a putative binding site near the head-neck region. The resulting model permitted the identification of two nonpeptidic entry inhibitors. Here, we present the design, synthesis, and bioevaluation of several series of fusion inhibitors and describe their structure-activity relationships (SAR). Five simply substituted anilides show low-microM blockade of the MV, one of which (AS-48) exhibits IC50 = 0.6-3.0 microM across a panel of wild-type MV strains found in the field. Molecular field topology analysis (MFTA), a 2D QSAR approach based on local molecular properties (atomic charges, hydrogen-bonding capacity and local lipophilicity), applied to the anilide series suggests structural modifications to improve potency.