A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination.

Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.

Identification of two hub genes and miRNA­mRNA interactions in chronic obstructive pulmonary disease (COPD) plasma.

BACKGROUND: We aimed to identify hub genes in chronic obstructive pulmonary disease (COPD) plasma through the exploration of a putative miRNA-mRNA regulatory network. METHODS: Three datasets (GSE24709, GSE102915, GSE136390) were utilized to discern differentially expressed miRNAs (DEMs) between COPD and normal plasma. miRNET was employed to predict the potential targets of DEMs. Subsequent GO and KEGG analyses were conducted using DAVID. For the construction of the protein-protein interaction (PPI) network and screening of hub genes, STRING and Cytoscape were employed. The expression validation was assessed through GSE56768. RESULTS: The results revealed 395 genes targeted by up-regulated DEMs and 234 genes targeted by down-regulated DEMs. The target genes exhibited significant enrichment in the PI3K-Akt signaling pathway and the p53 signaling pathway. Through the validation of hub genes' expression, we proposed two potential miRNA-mRNA interactions: miR-126-5p/miR-495-3p/miR-193b-3p - YWHAZ and miR-937-5p/miR-183-5p/miR-34c-5p/miR-98-5p/miR-525-3p/miR-215-5p - ACTB. CONCLUSIONS: In conclusion, our study posits potential miRNA-mRNA interactions in COPD by analyzing datasets from public databases, contributing valuable insights into the understanding of COPD pathogenesis and potential therapeutic avenues.

Clinical application of posterior tibial artery or peroneal artery perforator flap in curing plate exposure after ankle fracture fixation.

The study aims to evaluate the clinical application of posterior tibial artery or peroneal artery perforator flap in the treatment of plate exposure after ankle fracture fixation. A posterior tibial artery or peroneal artery perforator flap was used on 16 patients with plate exposure after ankle fracture fixation in our hospital between July 2018 and July 2021. The time required to harvest the flap, the amount of intraoperative blood loss, the duration of postoperative drainage tube placement, the outcome of the flap and the healing observed at the donor site are reported. The sizes of the flaps were 2.5-7.0 cm × 5.0-18.0 cm and averaged 4.0 cm × 12.0 cm. The time required to harvest the posterior tibial artery or peroneal artery perforator flap ranged from 35 to 55 min and averaged 45 min. The amount of intraoperative blood loss ranged from 20 to 50 mL and averaged 35 mL. The duration of postoperative drainage tube placement ranged from 3 to 5 days and averaged 4 days. A total of 15 flaps survived and one flap had partial necrosis and survived after conservative treatment. All donor area defects were directly sewed and stitched without complications. There are multiple advantages of the posterior tibial artery or peroneal artery perforator flap, including simple preparation technique, reliable repair of the defects and without the need for performing microvascular anastomosis. It can be safely used in curing plate exposure after ankle fracture fixation and worth popularizing in grassroots hospitals.

ARID1A downregulation promotes cell proliferation and migration of colon cancer via VIM activation and CDH1 suppression.

According to our prior findings, ARID1A expression is decreased in colon cancer, which has a poor prognosis. In this study, we investigated the ARID1A-VIM/CDH1 signalling axis's role in colon cancer proliferation and migration. The differentially expressed genes in cells that might be controlled by ARID1A were discovered by a database screening for ARID1A knockout. qPCR was used to analyse ARID1A and EMT markers expression levels in colon cancer. We utilized siRNA RID1A to explore the influence of ARID1A silencing on EMT in CRC cells. The function of ARID1A in the colon was investigated utilizing the wound healing, transwell and CCK-8 WST- assays. The molecular mechanism by which ARID1A regulates VIM and CDH1 was elucidated using chip-qPCR. Numerous genes involved in EMT were dysregulated in the absence of ARID1A. VIM expression increased in cells lacking ARID1A expression and vice versa. Many COAD samples with high ARID1A mRNA expression had low VIM mRNA expression, despite the relevance. CDH1 gene was positively correlated with ARID1A. Moreover, siRNA-ARID1A-transfected cells accelerated cell migration and invasion and increased cell proliferation rate in vitro. Chip-qPCR analysis showed that ARID1A binds to the promoters of both genes and changes their expression in colon cancer. ARID1A inactivation is associated with VIM activation and CDH1 suppression, which might serve as crucial molecules influencing COAD prognosis, accelerate tumour progression, and shorten patients' survival time, and promote metastases of COAD. Thus, depletion of ARID1A can be therapeutically exploited by targeting downstream effects to improve cancer treatment-related outcomes.

[Role of PTEN in FAK and α-SMA protein levels changes in human embryonic lung fibroblasts induced by silica].

OBJECTIVE: To investigate the roles of phosphatase and tensin homolog deleted on chromosome 10(PTEN) in focal adhesion kinase(FAK) and α-smooth muscle actin(α-SMA) protein levels changes in human embryonic lung fibroblasts(HELFs) induced by silica. METHODS: The HELF cells were cultured in low serum medium containing 0, 25, 50, 100 and 200 µg/cm~2 silica for 24 hours, and the cell counting kit-8(CCK8) experiment was used to determine the appropriate dose of silica for stimulation. Meanwhile, the effect of different doses of silica on the morphology of HELFs was observed under inverted microscope. 50 µg/cm~2 silica solution was used to culture HELFs for 0, 1, 2, 4, 8, 12 and 24 hours(h), and the control group were used as the control group. In addition, Western blot was used to detect HELFs PTEN, p-PTEN, FAK, p-FAK and α-SMA protein levels at each culture time. Besides, HELFs were cultured with 2×10~(-3) mol/L PTEN inhibitor(VO-Ohpic) and/or silica for 24 h, including HELFs group, HELFs plus silica group, and HELFs plus silica plus VO-Ohpic group, and the FAK, p-FAK and α-SMA in each group were also detected by Western blot. RESULTS: With the increase of silica dose, HELFs viability firstly increased and then decreased, and the cell viability of 50 µg/cm~2 group(144. 91±5. 10) was significantly higher than that of 0 µg/cm~2 group(101. 23±6. 57)(P<0. 05). Compared with the control group of silica treated HELFs, the expression levels of PTEN and p-PTEN at 12 h and 24 h were significantly decreased(PTEN: 0. 44±0. 08 at 12 h, 0. 25±0. 02 at 24 h; p-PTEN: 0. 09±0. 01 at 12 h, 0. 01±0. 00 at 24 h; all P values<0. 05); whereas, FAK at 12 h(0. 92±0. 05) and 24 h(0. 89±0. 01), and p-FAK(0. 77±0. 02) and α-SMA at 24 h(1. 32±0. 01) were significantly increased(all P values<0. 05). The expression levels of FAK(0. 25±0. 03), p-FAK(0. 40±0. 02) and α-SMA(0. 36±0. 01) of HELFs plus silica plus VO-Ohpic group were significantly higher than those of HELFs plus silica group(P<0. 05). CONCLUSION: While silica induces HELFs FAK, p-FAK, and α-SMA increase, PTEN may downregulate FAK, p-FAK and α-SMA expression levels.

[Effect of ERK/JNK cell signaling pathway in carbon black induced cytokine IL-6 and IL-8 expression changes].

OBJECTIVE: To investigate the roles of extracellular signal-regulated kinase(ERK)/c-Jun amino-terminal kinase(JNK) signaling pathway on the expression of interleukin-6(IL-6) and interleukin-8(IL-8) in human embryonic lung fibroblasts(HELF) induced by carbon black. METHODS: HELFs were cultured in RPMI-1640 medium containing 0, 15, 30, 60, 120 or 240 µg/mL carbon black for 24 h, and the appropriate dose of carbon black was determined by MTT assay result HELFs were divided into three groups: HELFs, HELFs transfected with ERK dominant negative mutant plasmid(DN-ERK) and HELFs transfected with JNK dominant negative mutant plasmid(DN-JNK). 100 µg/mL carbon black was used to treat HELFs(CB), DN-ERK HELFs(CB-DN-ERK), DN-JNK HELFs(CB-DN-JNK), and HELFs without any black carbon treatment were considered as control group. At 16 h after carbon black treatment, scanning electron microscope(SEM) was used to observe HELFs morphology and whether there were carbon black particless. At 0, 1, 2, 4, 8, 12, 24 and 36 h, the enzyme linked immunosorbent assay(ELISA) was used to detect CB and control groups HELFs IL-6 and IL-8 expression levels, whereas CB-DN-ERK and CB-DN-JNK HELFs were detected only at 24 h. RESULTS: SEM result showed no carbon black particles were observed in CB group HELFs, whereas their surface projections were increased. The CB group HELFs IL-6 expression levels at 2 h(44. 86±3. 65 ng/L) and 4 h(76. 52±3. 15 ng/L) were significantly lower than those of the control group(96. 78±2. 82 and 147. 32±3. 26 ng/L)(P<0. 05), whereas the IL-6 expression levels were significantly higher than those of the control group(105. 54±6. 10, 101. 27±5. 84 and 97. 15±5. 12 ng/L) at 16 h(202. 64±7. 20 ng/L), 24 h(200. 38±6. 20 ng/L) and 36 h(183. 54±4. 54 ng/L)(P<0. 001). At 24 h(136. 75±3. 81 ng/L) and 36 h(149. 12±2. 74 ng/L), the CB group IL-8 expression levels were significantly higher than those of the control group(75. 16±2. 84 and 73. 44±2. 15 ng/L)(P<0. 001). Compared with CB group HELFs, CB-DN-ERK and CB-DN-JNK groups HELFs had significantly lower IL-6 and IL-8 expression levels(P<0. 05). CONCLUSION: While carbon black induced HELFs IL-6 and IL-8 expression levels changes, ERK and JNK may upregulate IL-6 and IL-8 expression levels.

[Roles of ERK/JNK in carbon black induced AP-1 cell signaling pathway changes].

OBJECTIVE: To investigate the role of ERK/JNK in the alteration of activator protein-1(AP-1) signaling pathway in human embryonic lung fibroblasts(HELFs) induced by carbon black. METHODS: HELFs were cultured in RPMI 1640 medium containing 0, 15, 30, 60, 120 or 240 µg/mL carbon black for 24 h, and the appropriate dose of carbon black was determined by MTT assay result. HELFs were divided into three groups: HELFs, HELFs transfected with ERK dominant negative mutant plasmid(DN-ERK) and HELFs transfected with JNK dominant negative mutant plasmid(DN-JNK). 100 µg/mL carbon black was used to treat HELFs(CB), DN-ERK HELFs(CB-DN-ERK), DN-JNK HELFs(CB-DN-JNK), and HELFs without any treatment were considered as control group. At 0, 1, 2, 4, 8, 12, 24 and 36 h of CB and control groups HELFs, the western blot was used to detect ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels, and AP-1 activity was detected by luciferase method. Whereas CB-DN-ERK and CB-DN-JNK HELFs were detected only at 24 h. RESULTS: Compared with the protein expression levels at 0 h, CB group HELFs ERK and p-ERK protein expression increased at each time point, whereas p38 protein expression decreased. AP-1 activity of CB group HELFs was declined to the lowest at 8 h(0.72±0.12), and upregulated to the peak at 36 h(1.38±0.11). CB group HELFs c-Fos, p-c-Fos and c-Jun protein expression levels at each time point from 1 h to 24 h were greater than those of 0 h, and p-c-Jun protein expression levels at 1 h, 2 h, 4 h, 8 h, 36 h were also greater than those of 0 h. CB group HELFs AP-1 activity, ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels changes followed biphasic patterns. There were no statistically significant differences in AP-1 activity between CB group HELFs(1.03±0.10) and CB-DN-ERK group(1.02±0.04) or CB-DN-JNK group(1.09±0.10) HELFs(t=0.16, P=0.88; t=0.73, P=0.50). However, compared with CB group HELFs, c-Fos(t=5.31, P=0.01), p-c-Fos(t=4.33, P=0.01), p-c-Jun(t=10.95, P& lt; 0.01)in CB-DN-JNK group, and c-Fos protein expression levels in CB-DN-ERK group(t=42.72, P& lt; 0.01)were significantly decreased. CONCLUSION: While carbon black induces HELFs increased protein expression levels of ERK, p-ERK, c-Jun, p-c-Jun, c-Fos and p-c-Fos, JNK may upregulate c-Fos, p-c-Fos, p-c-Jun protein expression levels, and ERK may upregulate c-Fos protein expression level.

Nanopore-based Strategy for Selective Detection of Single Carcinoembryonic Antigen (CEA) Molecules.

Nanopores have become one of the most important tools for single-molecule sensing, but the challenge for selective detection of specific biomolecules still exists. In this contribution, we develop a new technique for sensing carcinoembryonic antigen (CEA), one of the important cancer biomarkers, using solid-state nanopores as a tool. The method is based on the specific affinity between aptamer (Apt) modified magnetic Fe3O4-Au nanoparticles (MNPs) and CEA, and the formed CEA-Apt-MNPs and remaining Apt-MNPs can transport the nanopores by applying a positive potential after magnetic separation. Due to the obvious particle size difference between CEA-Apt-MNPs and Apt-MPs, their corresponding blockage signals could be distinguished completely by the degree of the current decline. Moreover, the frequency of the blockage signals for CEA-Apt-MNPs is proportional to the concentration of CEA within certain limits, indicating that our designed nanopore sensing strategy can quantitatively detect CEA in complex samples. This work demonstrates that our designed nanopore-based strategy can be used for CEA sensing with good selectivity and sensitivity and also can be used to analyze other protein biomarkers for early diagnosis and monitoring of cancer, though the detection limit (0.6 ng/mL) is not relatively low. In future works, we plan to improve our detection limit by the improvement of the nanopipette preparation technology and detection method.

Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation.

Mechanism-based inactivation of l-aspartate-α-decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of ß-alanine from L-aspartic acid. In this study, a semi-rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site-saturation and C-terminal deletion, the variant Q5 (BsPanDI46V/I88M/K104S/I126* ) was generated. The catalytic half-life and the total turnover number (TTN) of Q5 were 3.48-fold and 2.52-fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 Å in Q0 vs. 5.5 Å in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l-aspartase (AspA) in a 15-L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L ß-alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism-based inactivation and boost catalytic stability.

Effect of MicroRNA-126a-3p on Bone Marrow Mesenchymal Stem Cells Repairing Blood-brain Barrier and Nerve Injury after Intracerebral Hemorrhage.

OBJECTIVE: Intracerebral hemorrhage (ICH) is a disease that threatens human health due to its high morbidity and mortality. On behalf of finding the better methods in the treatment of ICH, researchers pay more attention to a new technology which is finding effective genes to modify stem cells. METHODS: In this study, we isolated, cultured and identified bone marrow mesenchymal stem cells (MSCs) in vitro. Further, the MSCs (transfected with lentivirus expressing microRNA-126a-3p (miR-126)) were injected into the type Ⅶ collagenase-induced ICH rats to investigate the recovery effects of blood-brain barrier (BBB) and nerve damage in vivo. RESULTS: The MSCs surface marker molecules (CD29: 98.5%; CD90: 96.5%) were highly expressed, and the blood cell surface molecule was negatively expressed (CD45: 2%). Meanwhile, it was verified that miR-126 facilitated the differentiation of MSCs into vascular endothelial cells, owing to the rise of markers (CD31 and VE-cadherin). The modified neurological severity score, modified limb placing test score, brain water content and evans blue content were reduced after transplanted miR-126-modified MSCs. It was found that miR-126 accelerated the differentiation of MSCs into vascular endothelial cells via immunohistochemical staining in vivo. HE staining indicated the area of edema was obviously decreased compared with that in ICH + vector-MSCs group. MiR-126-modified MSCs alleviated the cell apoptosis in brain tissues by TUNEL assay. In addition, the mRNA and protein expression of protease activated receptor-1 and matrix metalloproteinase-9 were diminished, whilst the expression of zonula occludens-1 (ZO-1) and claudin-5 were enhanced in ICH+miR-126-MSCs group. Immunofluorescence assay revealed that miR-126-modified MSCs decreased the disruption of tight junction (ZO-1 and claudin-5). CONCLUSIONS: All data illustrate that miR-126-modified MSCs repair BBB and nerve injury after ICH.

STRIP2 silencing inhibits vascular smooth muscle cell proliferation and migration via P38-AKT-MMP-2 signaling pathway.

STRIP2 (FAM40B) was reported to regulate tumor cell migration. Our study aims to discuss the effect of STRIP2 in mouse aortic smooth muscle cell (MOVAS) proliferation and migration processes, which contributes greatly to atherosclerosis formation. In MOVAS cells, STRIP2 depletion suppressed cell proliferation and migration, which were related to a remarkable decrease in matrix metalloproteinases-2 (MMP-2)/MMP-9 expression. Additionally, P38 mitogen-activated protein kinases and Protein kinase B (AKT) are inactivated while extracellular signal-regulated kinase (ERK1/2) and jun N-terminal kinase (JNK) are activated upon STRIP2 silencing. SB203580 (P38 inhibitor) further reduced AKT phosphorylation (p-AKT) while dehydrocorydaline chloride (Dc; P38 activator) reversed this effect. Furthermore, Dc significantly recovered MMP-2 expression in STRIP2-knockdown cells. As expected, overexpressing STRIP2 exhibited a contrary effect. Dc and AKT activator SC79 reversed the inhibition of cell proliferation and migration induced by STRIP2 silencing. Interestingly, STRIP2 depletion increased vascular endothelial growth factor level significantly. Taken together, STRIP2 contributed to cell proliferation and migration through P38-AKT-MMP-2 signaling in MOVAS cells, indicating the importance of STRIP2 in atherosclerosis.

Susceptibility of Methicillin-Resistant Staphylococcus aureus to Five Quinazolinone Antibacterials.

The in vitro activities of five quinazolinone antibacterials, compounds Q1 to Q5, were tested against 210 strains of methicillin-resistant Staphylococcus aureus (MRSA). The MIC50/MIC90 values (in µg/ml) were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5. Several strains with high MIC values (from 8 to >32 µg/ml) for some of these compounds exhibited amino acid changes in the penicillin-binding proteins, which are targeted by these antibacterials.

Blockage of ROS and MAPKs-mediated inflammation via restoring SIRT1 by a new compound LF10 prevents type 1 diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a common and severe complication of diabetes. A multitude of factors are involved in the pathogenesis of DCM including chronic inflammation and oxidative stress. We have recently shown that compound LF10 prevents inflammatory responses in an animal model of lung injury. In the present study, we explored the protective effects and mechanism of LF10 against DCM using a mouse model of streptozotocin-induced diabetes and high glucose (HG)-challenged cultured cardiomyocytes. We show that LF10 suppressed diabetes-induced cardiomyocyte hypertrophy and fibrosis, which was accompanied by preservation of cardiac function in mice. Mechanistically, LF10 prevented increases in the levels of pro-inflammatory molecules and oxidative stress under in vitro and in vivo diabetic conditions. Moreover, LF10 restored HG-downregulated sirtuin 1 (SIRT1) in cardiomyocytes and prevented HG-induced activation of MAPKs. Using specific small-molecule regulators, we found that SIRT1 was an upstream signal of MAPKs. In conclusion, LF10 inhibited ROS and MAPKs-mediated inflammation by restoring SIRT1, and prevented development of DCM. LF10 targeted both oxidative stress and inflammation, two tightly interconnected pathogenic pathways, which makes LF10 a highly advantageous therapeutic drug potential.

[Role of p53/p21 signal transduction pathway in the malignant transformation of human embryonic lung fibroblasts induced by quartz].

OBJECTIVE: To observe the expression of p53 protein and investigate the roles of p53 in the expressions and interactions of p21, cyclin D1 and cyclin dependent kinase 4(CDK4) proteins in malignant transformation of human embryonic lung fibroblasts(T-HELF) induced by quartz. METHODS: The cytosolic protein and nuclear protein of both HELF and T-HELF cells were extracted by the separation technique of cytoplasm and nuclei. The distribution and expression of p53, phosphorylated p53 and p21 proteins were detected by Western blot. Based on the RNA interference technique, p53 siRNA was transfected into T-HELF cells to observe the protein expression and change of p53, phosphorylated p53, p21, cyclin D1 and CDK4, while the control group was conducted by transfecting the CMV-neo blank vector into the plasmid. The expression levels of p21, cyclin D1 and CDK4 protein complex in HELF and T-HELF cells were detected by immunoprecipitation. After adding 20 µmol/L of p53 chemical inhibitor pifithrin-α(PFT-α) and 2 µg of p53 siRNA into T-HELF cells respectively, the effect of p53 protein inhibition on p21, cyclin D1 and CDK4 protein complex was also observed. RESULTS: Quartz stimulation of HELF caused a significant increase in the expression of p53 and phosphorylated p53 protein in the nucleus(P<0. 05). The protein expression of p53 in the nucleus of T-HELF was significantly lower than that of HELF(P<0. 05). After transfection of p53 siRNA, the expression of p53 protein was decreased and the expression of p21 and cyclin D1 protein was increased compared with the control group(P<0. 05), while the change of expression in CDK4 was not observed(P>0. 05). Additionally, the result of immunoprecipitation showed that the inhibition of p53 expression could down-regulate the expression level of the binding complex between p21 and cyclin D1 protein(P<0. 05). However, this effect on p21-CDK4 and cyclin D1-CDK4 protein complex was not observed(P>0. 05). CONCLUSION: By regulating the expression and protein-protein interaction between p21 and cyclin D1, p53 would participate in quartz-induced malignant transformation.

Fibroblast growth factor 1 ameliorates diabetic nephropathy by an anti-inflammatory mechanism.

Inflammation plays a central role in the etiology of diabetic nephropathy, a global health issue. We observed a significant reduction in the renal expression of fibroblast growth factor 1, a known mitogen and insulin sensitizer, in patients with diabetic nephropathy and in mouse models implying that fibroblast growth factor 1 possesses beneficial anti-inflammatory and renoprotective activities in vivo. To test this possibility, we investigated the effects of chronic intraperitoneal administration of fibroblast growth factor 1 into both the streptozotocin-induced type 1 diabetes and db/db type 2 diabetes models. Indeed, recombinant fibroblast growth factor 1 significantly suppressed renal inflammation (i.e., cytokines, macrophage infiltration), glomerular and tubular damage, and renal dysfunction in both type 1 and type 2 diabetes mice. Fibroblast growth factor 1 was able to correct the elevated blood glucose levels in type 2 but not in type 1 diabetic mice, suggesting that the anti-inflammatory effect of fibroblast growth factor 1 was independent of its glucose-lowering activity. The mechanistic study demonstrated that fibroblast growth factor 1-mediated inhibition of the renal inflammation in vivo was accompanied by attenuation of the nuclear factor κB and c-Jun N-terminal kinase signaling pathways, further validated in vitro using cultured glomerular mesangial cells and podocytes. Thus, fibroblast growth factor 1 holds great promise for developing new treatments for diabetic nephropathy through countering inflammatory signaling cascades in injured renal tissue.

Engineering synergetic CO2-fixing pathways for malate production.

Increasing the microbial CO2-fixing efficiency often requires supplying sufficient ATP and redirecting carbon flux for the production of metabolites. However, addressing these two issues concurrently remains a challenge. Here, we present a combinational strategy based on a synergetic CO2-fixing pathway that combines an ATP-generating carboxylation reaction in the central metabolic pathway with the ATP-consuming RuBisCO shunt in the carbon fixation pathway. This strategy provides enough ATP to improve the efficiency of CO2 fixation and simultaneously rewires the CO2-fixing pathway to the central metabolic pathway for the biosynthesis of chemicals. We demonstrate the application of this strategy by increasing the CO2-fixing rate and malate production in the autotroph Synechococcus elongatus by 110% and to 260 µM respectively, as well as increasing these two factors in the heterotrophic CO2-fixing Escherichia coli by 870% and to 387 mM respectively.

7ß-Methyl substituent is a structural locus associated with activity cliff for nepenthone analogues.

With the purpose of identifying novel selective κ opioid receptor (KOR) antagonists as potential antidepressants from nepenthone analogues, starting from N-nor-N-cyclopropylmethyl-nepenthone (SLL-020ACP), a highly selective and potent KOR agonist, a series of 7ß-methyl-nepenthone analogues was conceived, synthesized and assayed on opioid receptors based on the concept of hybridization. According to the pharmacological results, the functional reversal observed in orvinol analogues by introduction of 7ß-methyl substituent could not be reproduced in nepenthone analogues. Alternatively, introduction of 7ß-methyl substituent was associated with substantial loss of both subtype selectivity and potency but not efficacy for nepenthone analogues, which was not found in 7ß-methyl orvinol analogues. Surprisingly, SLL-603, a 7ß-methyl analogue of SLL-020ACP, was identified to be a KOR full agonist. The possible molecular mechanism for the heterogeneity in activity cliff was also investigated. In conclusion, 7ß-methyl substituent was a structural locus associated with activity cliff and demonstrated as a pharmacological heterogeneity between nepenthone and orvinol analogues that warrants further investigations.

Inhibition of Epidermal Growth Factor Receptor (EGFR) Reduces Lipopolysaccharide (LPS)-Induced Activation and Inflammatory Cytokines in Hepatic Stellate Cells In Vitro.

BACKGROUND Epidermal growth factor receptor (EGFR) expression is associated with hepatic fibrogenesis. Activated hepatic stellate cells (HSCs) release inflammatory cytokines and extracellular matrix (ECM). The aim of this in vitro study was to investigate HSCs, activated by lipopolysaccharide (LPS), and the role of EGFR using the small molecule EGFR inhibitor, AG1478, and using knockdown of the EGFR gene using small interfering RNA (siRNA) cell transfection. MATERIAL AND METHODS HSCs, isolated from male Sprague-Dawley rats, were cultured and treated with and without LPS (100 ng/mL), with and without AG1478 (2.5 µM and 5.0 µM) Cell survival and proliferation were studied using an MTT assay. Western blot was used to measure levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IκBα, cytoplasm and nuclear NFκB and EGFR in the cell lysates before and after small interfering RNA (siRNA) transfection. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to measure the mRNA levels of transforming growth factor (TGF)-ß, Col-1, and α-smooth muscle actin (SMA). The Toll-like receptor 4 (TLR4) antagonist TAK-242 and the selective c-Src inhibitor, PP2 in LPS induced-EGFR phosphorylation was evaluated using Western blot. RESULTS Inhibition of EGFR decreased LPS-induced HSC proliferation and inflammatory cytokines. The TLR4 antagonist TAK-242, and the c-Src inhibitor, PP2 reduced EGFR activation of HSCs, indicating a possible role for the TLR4/c-Src signaling cascade in LPS-induced HSC activation. CONCLUSIONS Activation of HSCs by LPS in vitro, including the expression of inflammatory cytokines and mediators of fibrogenesis, were shown to be dependent on the expression of EGFR.

CRH promotes human colon cancer cell proliferation via IL-6/JAK2/STAT3 signaling pathway and VEGF-induced tumor angiogenesis.

Corticotrophin-releasing hormone (CRH) has been demonstrated to participate in various diseases. Our previous study showed that its receptor CRHR1 mediated the development of colitis-associated cancer in mouse model. However, the detailed mechanisms remain unclear. In this study, we explored the oncogenetic role of CRH/CRHR1 signaling in colon cancer cells. Cell proliferation and colony formation assays revealed that CRH contributed to cell proliferation. Moreover, tube formation assay showed that CRH-treated colon cancer cell supernatant significantly promoted tube formation of human umbilical vein endothelial cells (HUVECs). And these effects could be reversed by the CRHR1 specific antagonist Antalarmin. Further investigation showed that CRH significantly upregulated the expressions of interlukin-6 (IL-6) and vascular endothelial growth factor (VEGF) through activating nuclear factor-kappa B (NF-κB). The CRH-induced IL-6 promoted phosphorylation of janus kinase 2 (JAK2) and signal transducers and activators of transcription 3 (STAT3). STAT3 inhibition by Stattic significantly inhibited the CRH-induced cell proliferation. In addition, silence of VEGF resulted in declined tube formation induced by CRH. Taken together, CRH/CRHR1 signaling promoted human colon cancer cell proliferation via NF-κB/IL-6/JAK2/STAT3 signaling pathway and tumor angiogenesis via NF-κB/VEGF signaling pathway. Our results provide evidence to support a critical role for the CRH/CRHR1 signaling in colon cancer progression and suggest its potential utility as a new therapeutic target for colon cancer.

MD2 mediates angiotensin II-induced cardiac inflammation and remodeling via directly binding to Ang II and activating TLR4/NF-κB signaling pathway.

Angiotensin II (Ang II) induces cardiac inflammation and remodeling. Emerging evidence indicates that Ang II may utilize the Toll-like receptor 4 (TLR4) signaling pathway in mediating pro-inflammatory and pro-fibrotic activities. However, the precise mechanism is poorly understood. Myeloid differentiation 2 (MD2), a molecule that physically binds to TLR4, confers lipopolysaccharide responsiveness and may also be involved in mediating the actions of Ang II. We hypothesize that MD2 plays an essential role in cardiac inflammation and remodeling induced by local Ang II, and inhibition of MD2 can attenuate Ang II-induced cardiac dysfunction. Using a specific small molecule MD2 blocker L6H21 and the MD2 knockout mice, we show that MD2 deficiency significantly reduces cardiac inflammation and subsequent fibrosis, hypertrophy, and dysfunction in mice challenged with subcutaneous injection of Ang II. In rat cardiomyocyte-like H9c2 cells as well as rat primary cardiomyocytes, inhibition of MD2 by L6H21 or siRNA knockdown suppressed the Ang II-induced TLR4 signaling pathway activation including MyD88 recruitment, and reduced cardiomyocyte hypertrophy and matrix protein expression. These pro-inflammatory activities of Ang II were independent of the AT1 receptor. Finally, we demonstrated the direct interaction between Ang II and MD2 protein via hydrogen bonds on Arg-90, Glu-92, and Asp-100. Ang II produces an inflammatory response and cardiac remodeling by directly binding to MD2, activating MD2/TLR4 complex, and recruiting MyD88. MD2 may be a new therapeutic target for Ang II-mediated cardiac inflammation and remodeling.