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The anti-Clostridium difficile infection (CDI) drug fidaxomicin is a natural polyketide metabolite mainly produced by Micromonosporaceae, such as Actinoplanes deccanensis, Dactylosporangium aurantiacum, and Micromonospora echinospora. In the present study, we employed a stepwise strategy by combining heterologous expression, chassis construction, promoter engineering, activator and transporters overexpression, and optimization of fermentation media for high-level production of fidaxomicin. The maximum yield of 384 mg/L fidaxomicin was achieved with engineered Streptomyces albus D7-VHb in 5 L-tank bioreactor, and it was approximately 15-fold higher than the native strain Actinoplanes deccanensis YP-1 with higher strain stability and growth rate. This study developed an enhanced chassis strain, and for the first time, achieved the heterologous synthesis of fidaxomicin through a combinatorial metabolic engineering strategy.

Abstract Background Oritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production. Results Firstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities. Conclusion Under approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.

ABSTRACT Fidaxomicin is an 18-membered glycosidic macrolide polyketide that was clinically used in the treatment of Clostridium difficile infection in 2011. Although the biosynthetic mechanism and pathway-specific regulator of fidaxomicin were well elaborated, little is known about the pleiotropic regulators outside the fidaxomicin gene cluster. To further reveal the transcriptional regulatory mechanism of fidaxomicin biosynthesis in Actinoplanes deccanensis YP-1, we used the 5′-biotin-labeled fadR1 promoter (fadS5R1p) as a probe to affinity isolate the fadS5R1p-interactive protein MtrA, a response regulator of the two-component system MtrAB, from the proteome of mycelia. MtrA is bound directly to fadS5R1p to positively regulate gene cluster expression and fidaxomicin production. Fidaxomicin production was improved by 37% in the mtrA overexpressed strain. Furthermore, we revealed that MtrA tends to bind GTKAYS motifs and influence genes involved in secondary metabolites, phosphate transport, methionine transport, nitrogen metabolism, drug transport, membrane properties, etc. Overall, for the first time at the transcriptional level, we comprehensively revealed a pleiotropic regulator, MtrA, that not only directly mediates the FadR1-specific pathway to regulate the proper production of fidaxomicin but also participates in bacterial growth and development. IMPORTANCE Cascade regulation networks are almost present in various kinds of microorganisms, but locating and systematically elucidating specific pleiotropic regulators related to a certain gene cluster can be a tricky problem. Here, based on the promoter of the fidaxomicin pathway-specific regulator FadR1, we utilized a “DNA to Proteins” affinity purification method and captured a global regulator MtrA, which positively regulates fidaxomicin biosynthesis. In the mtrA overexpressed strain, the production of fidaxomicin was improved by 37% compared to the native strain. Then, we combined the “Protein to DNAs” affinity purification method (DAP-seq) with the results of RNA-seq and systematically elucidated the primary and secondary metabolic processes in which MtrA directly or indirectly participates. Thus, our work brought up a new way to improve fidaxomicin production from the perspective of global regulation and analyzed the regulatory mechanism of MtrA. Meanwhile, we provided a novel methodology for the research of cascade regulation networks and vital secondary metabolites.

Objective Based on previous studies on a class of tumor-targeted indocyanine small molecules, we aimed to synthesize a new analog modified with water-soluble polyethylene glycol (PEG) and then investigated its physicochemical properties and role in tumor targeting photodynamic therapy. Methods PEG-modified indocyanine small molecules were designed and synthesized by using 2, 3, 3-trimethylindole and polyethylene glycol monomethyl ether as raw materials. Its chemical structure was characterized by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS); its physicochemical properties and cancer targeting photodynamic effect were investigated by determining its water solubility, optical spectra, cell uptake, cell viability, singlet oxygen, and reactive oxygen production. Results Based on a converging strategy of synthesis and through 6 steps of chemical reactions, a PEG-modified indocyanine small molecule (PEG-808-NM2) was successfully produced. Its structure was confirmed by NMR spectroscopy and MS. Compared to 808-NM2, PEG-808-NM2 significantly increased water solubility and exhibited excellent near-infrared fluorescence property in both methanol and water solution. PEG-808-NM2 preferentially accumulated in RENCA cells (a mouse renal adenocarcinoma cell line) rather than HK-2 cells (a tubular cell line from normal human kidneys). Under 808 nm-laser irradiation, PEG-808-NM2 induced a great amount of singlet oxygen and intracellular reactive oxygen species. Conclusion Our synthesized PEG-modified indocyanine small molecules not only keep the cancer preferential accumulation and the role in near-infrared imaging as original indocyanine, but also obtain significantly improved physicochemical properties and photosensitizing effect. They exert significant photodynamic therapeutic effect on renal cell carcinoma cells.

Abstract Background Large-scale genome reduction has been performed to significantly improve the performance of microbial chassis. Identification of the essential or dispensable genes is pivotal for genome reduction to avoid synthetic lethality. Here, taking Streptomyces as an example, we developed a combinatorial strategy for systematic identification of large and dispensable genomic regions in Streptomyces based on multi-omics approaches. Results Phylogenetic tree analysis revealed that the model strains including S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 were preferred reference for comparative analysis of candidate genomes. Multiple genome alignment suggested that the Streptomyces genomes embodied highly conserved core region and variable sub-telomeric regions, and may present symmetric or asymmetric structure. Pan-genome and functional genome analyses showed that most conserved genes responsible for the fundamental functions of cell viability were concentrated in the core region and the vast majority of abundant genes were dispersed in the sub-telomeric regions. These results suggested that large-scale deletion can be performed in sub-telomeric regions to greatly streamline the Streptomyces genomes for developing versatile chassis. Conclusions The integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to generate more miscellaneous and versatile chassis with minimized genome for drug discovery.

Objective: This study was conducted for investigating the functions of circular RNA circRNA_100146 (circRNA_100146) in the development of prostate cancer (PCa) and identifying the underlying mechanisms of the circRNA_100146/miR-615-5p/TRIP13 axis.Materials and Methods: Under the support of RT-PCR, the expression of circRNA_100146 in PCa cells was examined. Cell Counting Kit-8 (CCK-8) assays and clone formation assays were applied to the assessment of cell proliferation. We then determined cell invasion and migration through transwell assays and wound healing assays. RNA pull-down assays and luciferase reporter assays were performed for the exploration of the regulatory effects of potential molecules on the expressions of the targeting genes. In addition, a nude mouse xenograft model was applied to demonstrate the oncogenic roles of circRNA_100146 in PCa.Results: CircRNA_100146 expression was distinctly upregulated in PCa cells. Silencing of circRNA_100146 suppressed PCa cells’ invasion, migration, and proliferation. CircRNA_100146 sponged miR-615-5p to suppress its expressions, while miR-615-5p targeted the 3’-UTR of TRIP13 to repress the expression of TRIP13. In addition, we observed that knockdown of miR-615-5p reversed the suppression of circRNA_100146 silence on the proliferation and invasion of PCa cells. In addition, the tumor growth was also suppressed by silencing circRNA_100146 in vivo.Conclusion: CircRNA_100146 is a tumor promoter in PCa, which promoted progression by mediating the miR-615-5p/TRIP13. CircRNA_100146 can be a potential candidate for targeted therapy of PCa.

C19H21NO2S, orthorhombic, Pbca (no. 61), a = 16.792(5) Å, b = 9.153(3) Å, c = 22.809(6) Å, V = 3505.8(16) Å3, Z = 8, Rgt(F) = 0.0622, wRref(F2) = 0.1494, T = 296(2) K.

C24H23FN2O4S, orthorhombic, Pbca (no. 19), a = 14.3646(18) Å, b = 15.5927(19) Å, c = 19.645(3) Å, V = 4400.1(10) Å3, Z = 8, Rgt(F) = 0.0349, wRref(F2) = 0.0982, T = 296(2) K.

C25H25NO2S, monoclinic, P21 (no. 4), a = 9.371(4) Å, b = 11.397(4) Å, c = 9.777(4) Å, β = 95.117(6)°, V = 1040.0(7) Å3, Z = 2, Rgt(F) = 0.0423, wRref(F2) = 0.0921, T = 296(2) K.

The next-generation wireless sensor network (WSN) has the capability of carrying hundreds of high-definition video streams, beside the feature of massive employment of energy-efficient nodes. However, several challenges are identified with respect to the video bearing, such as the different video formats, enormous size of “raw” video, and compatibility with heterogeneous terminal devices. The video transcoding system (VTS) is widely believed to address these challenges. This paper introduces a cloud-based, more specifically, Hadoop-based, video transcoding system to fulfill the vision of bearing hundreds of HD video streams in the next generation WSN, with a discussion on optimization of several significant parameters. This paper obtains three remarkable results: (1) there is an optimal value of the number of Mappers; (2) the optimal value is closely related to the file size; (3) the transcoding time depends principally on the duration of video files rather than their sizes.

Radionuclide uranium has both a chemical and radioactive toxicity, leading to severe nephrotoxicity as it predominantly deposits itself in the kidneys after entering into human bodies. It crosses renal cell membranes, accumulates in mitochondria and causes mitochondrial oxidative damage and dysfunction. In this study, a mitochondria-targeted heptamethine indocyanine small molecule chelator modified with gallic acid (IR-82) is synthesized for uranium detoxication. Both gallic acid and sulfonic acid, as two hydrophilic endings, make IR-82, being excreted feasibly through kidneys. Gallic acid with polyphenol groups has a steady metal chelation effect and potent antioxidant ability, which may facilitate IR-82-alleviated uranium nephrotoxicity simultaneously by enhancing uranium decorporation from the kidneys and reducing mitochondrial oxidative damage. Cell viability assays demonstrate that IR-82 can significantly improve the cell viability of uranium-exposed human renal (HK-2) cells. It is also demonstrated to accumulate in mitochondria and reduce mitochondrial ROS and total intracellular ROS, as well as intracellular uranium content. In vivo imaging experiments in mice show that IR-82 could be excreted out through kidneys. ICP-MS tests further reveal that IR-82 can efficiently decrease the uranium deposition in mouse kidneys. IR-82 treatment improves the animal survival rate and renal function of experimental mice after high-dose uranium exposure. Collectively, our study may evidence that the development of uranium decorporation agents with kidney–mitochondrion dual targeting abilities is a promising strategy for attenuating uranium-induced nephrotoxicity. [ABSTRACT FROM AUTHOR]

Background: Fat overload syndrome is a rare and severe adverse reaction triggered by the infusion of a single source of lipid emulsion, resulting in elevated blood triacylglycerol (TG) levels. The majority of literature reports focus on cases of fat overload syndrome in patients with mild symptoms. This case is significant because it demonstrates the diagnostic and therapeutic experience and provide valuable insights for the management for severe fat overload syndrome. Case Presentation: We present a case report of a female patient who developed fat overload syndrome following prolonged and excessive infusion of lipid emulsion after colon resection surgery. In the setting of compromised immune function and malnutrition, the patient's pulmonary infection and respiratory distress symptoms have further exacerbated. Hence, in addition to severe pancreatitis, the patient has also contracted severe pneumonia. Upon admission, tracheal intubation, plasma exchange and blood perfusion were performed. Subsequently, comprehensive treatment was provided, including anti-infection, antispasmodic, acid suppression, enzyme inhibition, as well as targeted supportive measures to stabilize electrolytes and nutritional status. After treatment, there was a progressive reduction in blood lipid levels. After assessing the relevant risks, it was deemed necessary to perform an emergency computed tomography (CT)-guided percutaneous drainage tube placement procedure targeting the necrotic area of the pancreas while the patient was still intubated. Finally, the patient was discharged from the hospital. Conclusion: The case highlights the association between fat overload syndrome and pancreatitis as well as the use of lipid emulsions and suggests the treatment strategies for severe fat overload syndrome. [ABSTRACT FROM AUTHOR]