A light-controlled cell lysis system in bacteria

Journal of Industrial Microbiology & Biotechnology - Intracellular products (e.g., insulin), which are obtained through cell lysis, take up a big share of the biotech industry. It is often...     

Early treatment with fumagillin, an inhibitor of methionine aminopeptidase-2, prevents Pulmonary Hypertension in monocrotaline-injured rats

Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2) regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC) with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients.     

Copper in organic proteinate or inorganic sulfate form is equally bioavailable for broiler chicks fed a conventional corn-soybean meal diet

An experiment was conducted to investigate the bioavailability of organic copper (Cu) proteinate relative to inorganic Cu sulfate for broiler chicks fed a conventional corn-soybean meal basal diet. A total of 320 day-old Arbor Acres commercial male chicks were assigned to one of five treatments in a completely randomized design involving a 2 × 2 factorial arrangement with two levels of added Cu (125 or 250 mg Cu/kg) and two Cu sources (Cu proteinate and Cu sulfate) plus a control with no added Cu for an experimental phase of 42 days. Plasma and liver tissue samples were collected at both 21 and 42 days of age, and bile samples were also obtained at 42 days of age for Cu analyses. The Cu concentrations in liver and bile increased linearly (P < 0.001) on both days 21 and 42 as dietary Cu levels increased. No significant (P > 0.17) linear regression relationships were observed between plasma Cu concentrations on days 21 and 42 or log10 liver Cu concentration on day 21 and daily analyzed Cu intake. Therefore, based on the slope ratios from multiple linear regressions of log10 liver and bile Cu concentrations with daily analyzed Cu intake on day 42, when Cu sulfate was set as 100%, the estimated relative bioavailability values of Cu proteinate were 78.8% for log10 liver Cu concentration and 79.3% for log10 bile Cu concentration, respectively. There was no significant (P > 0.08) difference in bioavailability between Cu proteinate and Cu sulfate for broilers chicks in this experiment.     

Mining and engineering exporters for titer improvement of macrolide biopesticides in Streptomyces

Exporter engineering is a promising strategy to construct high-yield Streptomyces for natural product pharmaceuticals in industrial biotechnology. However, available exporters are scarce, due to the limited knowledge of bacterial transporters. Here, we built a workflow for exporter mining and devised a tunable plug-and-play exporter (TuPPE) module to improve the production of macrolide biopesticides in Streptomyces. Combining genome analyses and experimental confirmations, we found three ATP-binding cassette transporters that contribute to milbemycin production in Streptomyces bingchenggensis. We then optimized the expression level of target exporters for milbemycin titer optimization by designing a TuPPE module with replaceable promoters and ribosome binding sites. Finally, broader applications of the TuPPE module were implemented in industrial S. bingchenggensis BC04, Streptomyces avermitilis NEAU12 and Streptomyces cyaneogriseus NMWT1, which led to optimal titer improvement of milbemycin A3/A4, avermectin B_1a and nemadectin α by 24.2%, 53.0% and 41.0%, respectively. Our work provides useful exporters and a convenient TuPPE module for titer improvement of macrolide biopesticides in Streptomyces. More importantly, the feasible exporter mining workflow developed here might shed light on widespread applications of exporter engineering in Streptomyces to boost the production of other secondary metabolites.     

Manganese elevates manganese superoxide dismutase protein level through protein kinase C and protein tyrosine kinase

Three experiments were conducted to investigate the effects of inorganic and organic Mn sources on MnSOD mRNA, protein and enzymatic activity and the possible signal pathways. The primary broiler myocardial cells were treated with MnCl₂ (I) or one of organic chelates of Mn and amino acids with weak, moderate (M) or strong (S) chelation strength for 12 and 48 h. Cells were preincubated with superoxide radical anions scavenger N-acetylcysteine (NAC) or specific inhibitors for MAPKs and protein tyrosine kinase (PTK) or protein kinase C (PKC) for 30 min before treatments of I and M. The MnSOD mRNA, protein and enzymatic activity, phosphorylated MAPKs or protein kinases activations were examined. The results showed that additions of Mn increased (P < 0.05) MnSOD mRNA levels and M was more effective than I. Additions of Mn elevated (P < 0.05) MnSOD protein levels and enzymatic activities, and no differences were found among I and M. Addition of NAC did not decrease (P > 0.05) Mn-induced MnSOD mRNA and protein levels. None of the three MAPKs was phosphorylated (P > 0.05) by Mn. Additions of Mn decreased (P < 0.05) the PTK activities and increased (P < 0.05) the membrane PKC contents. Inhibitors for PTK or PKC decreased (P < 0.05) Mn-induced MnSOD protein levels. The results suggested that Mn-induced MnSOD mRNA and protein expressions be not related with NAC, and MAPK pathways might not involve in Mn-induced MnSOD mRNA expression. PKC and PTK mediated the Mn-induced MnSOD protein expression.     

Glycogen synthase kinase 3 (GSK3)-inhibitor SB216763 promotes the conversion of human umbilical cord mesenchymal stem cells into neural precursors in adherent culture

The ability to generate neural progenitor cells from human umbilical cord mesenchymal stem cells (hUC-MSCs) has provided an option to treat neurodegenerative diseases. To establish a method for this purpose, we characterized the early neural markers of hUC-MSCs-derived cells under different conditions. We found that neither the elimination of signals for alternative fate nor N2 supplement was sufficient to differentiate hUC-MSCs into neural precursor cells, but the GSK3 inhibitor SB216763 could promote an efficient neural commitment of hUC-MSCs. The results indicated that Wnt/β-catenin might play an important role during the early neural differentiation of hUC-MSCs. Here, we report a method for hUC-MSCs to commit efficiently into a neural fate within a short period of time. This protocol provides an efficient method for hUC-MSCs-based neural regeneration.     

Coordinated regulation of the ribosome and proteasome by PRMT1 in the maintenance of neural stemness in cancer cells and neural stem cells

Previous studies suggested that cancer cells resemble neural stem/progenitor cells in regulatory network, tumorigenicity, and differentiation potential, and that neural stemness might represent the ground or basal state of differentiation and tumorigenicity. The neural ground state is reflected in the upregulation and enrichment of basic cell machineries and developmental programs, such as cell cycle, ribosomes, proteasomes, and epigenetic factors, in cancers and in embryonic neural or neural stem cells. However, how these machineries are concertedly regulated is unclear. Here, we show that loss of neural stemness in cancer or neural stem cells via muscle-like differentiation or neuronal differentiation, respectively, caused downregulation of ribosome and proteasome components and major epigenetic factors, including PRMT1, EZH2, and LSD1. Furthermore, inhibition of PRMT1, an oncoprotein that is enriched in neural cells during embryogenesis, caused neuronal-like differentiation, downregulation of a similar set of proteins downregulated by differentiation, and alteration of subcellular distribution of ribosome and proteasome components. By contrast, PRMT1 overexpression led to an upregulation of these proteins. PRMT1 interacted with these components and protected them from degradation via recruitment of the deubiquitinase USP7, also known to promote cancer and enriched in embryonic neural cells, thereby maintaining a high level of epigenetic factors that maintain neural stemness, such as EZH2 and LSD1. Taken together, our data indicate that PRMT1 inhibition resulted in repression of cell tumorigenicity. We conclude that PRMT1 coordinates ribosome and proteasome activity to match the needs for high production and homeostasis of proteins that maintain stemness in cancer and neural stem cells.     

Enantioseparation of chiral pharmaceuticals by vancomycin‐bonded stationary phase and analysis of chiral recognition mechanism

The drug chirality is attracting increasing attention because of different biological activities, metabolic pathways, and toxicities of chiral enantiomers. The chiral separation has been a great challenge. Optimized high‐performance liquid chromatography (HPLC) methods based on vancomycin chiral stationary phase (CSP) were developed for the enantioseparation of propranolol, atenolol, metoprolol, venlafaxine, fluoxetine, and amlodipine. The retention and enantioseparation properties of these analytes were investigated in the variety of mobile phase additives, flow rate, and column temperature. As a result, the optimal chromatographic condition was achieved using methanol as a main mobile phase with triethylamine (TEA) and glacial acetic acid (HOAc) added as modifiers in a volume ratio of 0.01% at a flow rate of 0.3 mL/minute and at a column temperature of 5°C. The thermodynamic parameters (eg, ΔH, ΔΔH, and ΔΔS) from linear van 't Hoff plots revealed that the retention of investigated pharmaceuticals on vancomycin CSP was an exothermic process. The nonlinear behavior of lnk′ against 1/T for propranolol, atenolol, and metoprolol suggested the presence of multiple binding mechanisms for these analytes on CSP with variation of temperature. The simulated interaction processes between vancomycin and pharmaceutical enantiomers using molecular docking technique and binding energy calculations indicated that the calculated magnitudes of steady combination energy (ΔG) coincided with experimental elution order for most of these enantiomers.     

Neuroprotective effects of ProBeptigen/CMI-168 on aging-induced cognitive decline and neuroinflammation in mice: a comparison with essence of chicken

Neuroinflammation and cognitive decline are the key pathological features in aging that bring detrimental impacts upon quality of life.However,there is no effec...