A modular toolkit for environmental Rhodococcus, Gordonia, and Nocardia enables complex metabolic manipulation.

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as Escherichia coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208, and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus, Nocardia, and Gordonia. This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux toward the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens. Rhodococcus, Gordonia, and Nocardia are three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these, Rhodococcus ruber C208 was shown to degrade polyethylene; Gordonia paraffinivorans can assimilate carbon from solid hydrocarbons; and Nocardia neocaledoniensis (and many other Nocardia spp.) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.

Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway.

Cytochrome P450 enzymes (CYP450s) exert mighty catalytic actions in cellular metabolism and detoxication, which play pivotal roles in cell fate determination. Preliminary data shows differential expression levels of CYP27C1, one of the "orphan P450s" in human lung cancer cell lines. Here, we study the functions of CYP27C1 in lung cancer progression and drug endurance, and explore its potential to be a diagnostic and therapeutic target for lung cancer management. Quantitative real-time PCR and immunoblot assays were conducted to estimate the transcription and protein expression level of CYP27C1 in human lung cancer cell lines, which was relatively higher in A549 and H1975 cells, but was lower in H460 cells. Stable CYP27C1-knockdown A549 and H1975 cell lines were established, in which these cells showed enhancement in cell proliferation, colony formation, and migration. In addition, aberrant IGF-1R/Akt/p53 signal transduction was also detected in stable CYP27C1-knockdown human lung cancer cells, which exhibited greater tolerance towards the treatments of anticancer agents (including vinorelbine, picropodophyllin, pacritinib, and SKLB610). This work, for the first time, reveals that CYP27C1 impacts lung cancer cell development by participating in the regulation of the IGF-1R/Akt/p53 signaling pathway, and the level of CYP27C1 plays indispensable roles in dictating the cellular sensitivity towards multiple anticancer agents.

Effects of CYP3A43 Expression on Cell Proliferation and Migration of Lung Adenocarcinoma and Its Clinical Significance.

The cytochrome P450s (CYP450s) include key oxidative enzymes involved in the metabolism of various carcinogens and anticancer drugs. Bioinformatic studies have demonstrated the association of CYP3A43 with liver cancer and ovarian cancer. However, the biological function of CYP3A43 in tumor progression remains unclear. To further reveal the role of CYP3A43 in tumor progression, we first analyzed the data from the UALCAN database and found that CYP3A43 was negatively correlated to the cancer staging and lymph node metastasis of lung adenocarcinoma (LUAD). We established stable CYP3A43-knockdown LUAD H1299 cell line and found that its knockdown enhanced cell proliferation, colony formation, and migration in vitro, and promoted the growth of tumor xenograft in vivo. Interestingly, when CYP3A43 was ectopically-expressed in the LUAD cell lines, decreased cell proliferation and ERK1/2 phosphorylation level were observed. Lastly, we also identified CYP3A43 co-expressed genes in LUAD from LinkedOmics database followed by GO and KEGG analyses. In conclusion, our results indicate the unprecedented role of CYP3A43 in the suppression of LUAD and provide new possibilities for targeted therapy of this life-threatening disease.

Phytofabrication of Nanoparticles as Novel Drugs for Anticancer Applications.

Cancer is one of the foremost causes of death globally and also the major stumbling block of increasing life expectancy. Although the primary treatment of surgical resection, chemotherapy, and radiotherapy have greatly reduced the mortality of cancer, the survival rate is still low because of the metastasis of tumor, a range of adverse drug reactions, and drug resistance. For all this, it is relevant to mention that a growing amount of research has shown the anticarcinogenic effect of phytochemicals which can modulate the molecular pathways and cellular events include apoptosis, cell proliferation, migration, and invasion. However, their pharmacological potential is hindered by their low water solubility, low stability, poor absorption, and rapid metabolism. In this scenario, the development of nanotechnology has created novel formulations to maximize the potential use of phytochemicals in anticancer treatment. Nanocarriers can enhance the solubility and stability of phytochemicals, prolong their half-life in blood and even achieve site-targeting delivery. This review summarizes the advances in utilizing nanoparticles in cancer therapy. In particular, we introduce several applications of nanoparticles combined with apigenin, resveratrol, curcumin, epigallocatechin-3-gallate, 6-gingerol, and quercetin in cancer treatment.

Interrogating the Function of Bicistronic Translational Control Elements to Improve Consistency of Gene Expression.

Context independent gene expression is required for genetic circuits to maintain consistent and predicable behavior. Previous efforts to develop context independent translation have leveraged the helicase activity of translating ribosomes via bicistronic design translational control elements (BCDs) located within an efficiently translated leader peptide. We have developed a series of bicistronic translational control elements with strengths that span several orders of magnitude, maintain consistent expression levels across diverse sequence contexts, and are agnostic to common ligation sequences used in modular cloning systems. We have used this series of BCDs to investigate several features of this design, including the spacing of the start and stop codons, the nucleotide identity upstream of the start codon, and factors affecting translation of the leader peptide. To demonstrate the flexibility of this architecture and their value as a generic modular expression control cassette for synthetic biology, we have developed a set of robust BCDs for use in several Rhodococcus species.

ACC2 is under-expressed in lung adenocarcinoma and predicts poor clinical outcomes.

PURPOSE: Acetyl-CoA Carboxylases (ACCs) are key fatty acid metabolic enzymes responsible for catalyzing the carboxylation of acetyl-CoA to malonyl-CoA. The role of ACC1 has been associated with tumor biology, but the role of ACC2 in cancer remains largely uncharacterized. METHODS: We conducted a transcriptomic analysis using GEPIA and Oncomine to study the expression of ACC2 in different cancers. Immunohistochemistry was used to examine the expression of ACC2 in lung cancer tissue microarray, and the correlation between ACC2 expression and clinical parameters was analyzed. Following ACC2 knockdown by RNA interference in A549 and HCC827 cells, Cell Counting Kit­8 and transwell assays were used to detect cell proliferation and migration. Real-time PCR was used to detect cell cycle-related genes in A549 cells. GEO dataset and KM-plotter database were used to analyze the relationship between ACC2 expression and the prognosis in lung cancer patients. RESULTS: We found that ACC2 is under-expressed in cancerous tissue and the expression of ACC2 is negatively correlated with tumor size, regional lymph-node metastases, and clinical stage of lung adenocarcinoma patients. In addition, knocking down ACC2 in A549 cells and HCC827 cells can promote cell proliferation and migration, and cell cycle-related genes MAD2L1 and CCNB2 were up-regulated after ACC2 was knockdown in A549 cells. Finally, we found that lung adenocarcinoma patients with under-expressed ACC2 have a worse prognosis. CONCLUSIONS: Our results suggest that ACC2 is a potential diagnostic and prognostic marker that negatively correlated with clinical outcomes in lung adenocarcinoma.

A postpartum enriched environment rescues impaired cognition and oxidative markers in aged mice with gestational inflammation.

INTRODUCTION: Previous studies have shown that gestational inflammation can accelerate age-associated cognitive decline (AACD) in maternal mice; enriched environments (EEs) have been reported to protect normally aging mice from AACD and improve mitochondrial function. However, it is unclear whether the nitrosative stress-related proteins tet methylcytosine dioxygenase 1 (TET1) and S-nitrosoglutathione reductase (GSNOR) are involved in the accelerated aging process of gestational inflammation and whether EEs can slow this process. METHODS: In this study, CD-1 female mice on the 15th day of pregnancy were injected with bacterial lipopolysaccharide (50 µg/kg; LPS group) or an equivalent amount of normal saline (CON group) from the abdominal cavity for 4 consecutive days. Twenty-one days after delivery, half of the LPS-treated mice were randomly selected for EE until the end of the behavioral experiment (LPS-E group). When the female rats were raised to 6 months and 18 months of age, the Morris water maze (MWM) was used to detect spatial learning and memory ability; RT-PCR and Western blots were used to measure the mRNA and protein levels of hippocampal TET1 and GSNOR. RESULTS: As for the control group, compared with 6-month-old mice, the spatial learning and memory ability of 18-month-old mice decreased, and the hippocampal TET1 and GSNOR mRNA and protein levels were decreased. Gestational inflammation exacerbated these age-related changes, but an EE alleviated the effects. Pearson's correlation analysis indicated that performance during the learning and memory periods in the MWM correlated with the levels of hippocampal TET1 and GSNOR. CONCLUSIONS: Our findings suggest that gestational inflammation accelerates age-related learning and memory impairments and that postpartum EE exposure could alleviate these changes. These effects may be related to hippocampal TET1 and GSNOR expression.

Transcriptional upregulation of MAPK15 by NF-κB signaling boosts the efficacy of combination therapy with cisplatin and TNF-α.

The efficacy of cisplatin in treating advanced non-small cell lung cancer is limited mainly because of insensitivity and/or acquired resistance. MAPK15, previously shown by us to enhance the sensitivity of the anti-cancer drug arsenic trioxide, could also enhance the sensitivity of other anti-cancer drugs. Here, we explore the potential role of MAPK15 in chemosensitivity to cisplatin in human lung cancer cells. Our results indicated that the expression level of MAPK15 was positively correlated with cisplatin sensitivity through affecting the DNA repair capacity of cisplatin-treated cells. The expression of MAPK15 was transcriptionally regulated by the TNF-α-activated NF-κB signaling pathway, and TNF-α synergized with cisplatin, in a MAPK15-dependent manner, to exert cytotoxicity in vitro and in vivo. Therefore, levels of TNF-α dictate the responsiveness/sensitivity of lung cancer cells to cisplatin by transcriptionally upregulating MAPK15 to enhance chemosensitivity, suggesting manipulation of MAPK15 as a strategy to improve the therapeutic efficacy of chemotherapeutic drugs.

Predictive Value of Red Blood Cell Distribution Width in Poststroke Depression.

PURPOSE: Red blood cell distribution width (RDW) is increased in a variety of inflammatory-related diseases. However, there is no report of its clinical significance in poststroke depression (PSD). This study explores the clinical significance of RDW in PSD patients. METHODS: A total of 185 patients with first-ever acute ischaemic stroke (AIS) in the Second Hospital of Anhui Medical University were chosen as subjects. A retrospective observational study was conducted from February 2019 to February 2020. PSD patients were diagnosed at 6 months after stroke based on the Diagnostic and Statistical Manual of Mental Disorders-IV criteria. Clinical and laboratory data were obtained from all patients. Coefficient of Variation (RDW-CV) and standard deviation (RDW-SD) were used to statistically report the performance of red blood cell distribution width. RESULTS: At the 6-month follow-up, 46 patients were diagnosed with PSD. Compared with non-PSD patients, PSD patients exhibited an increase in RDW-CV and RDW-SD, which positively correlated with serum interleukin 6 (IL-6) concentrations. In PSD patients, only RDW-SD demonstrated a consistent positive association with the Hamilton Rating Scale for Depression (HAM-D) scores at 6 months after admission. RDW-CV, RDW-SD, and IL-6 were recognized as independent predictors of PSD. The area under the receiver operating characteristic (ROC) curve (AUC) of RDW-SD was 0.796 (95% CI: 0.731-0.852) for the prediction of PSD, which was superior to that of RDW-CV. The specificity for predicting PSD was 60.43%, and the sensitivity was 91.30% if RDW-SD was higher than 43.80 fL. CONCLUSIONS: RDW-SD is a simple, inexpensive, rapid, and easily accessible parameter that can be used to predict PSD in patients with stroke.

Recent Progress of Nanocarrier-Based Therapy for Solid Malignancies.

Conventional chemotherapy is still an important option of cancer treatment, but it has poor cell selectivity, severe side effects, and drug resistance. Utilizing nanoparticles (NPs) to improve the therapeutic effect of chemotherapeutic drugs has been highlighted in recent years. Nanotechnology dramatically changed the face of oncology by high loading capacity, less toxicity, targeted delivery of drugs, increased uptake to target sites, and optimized pharmacokinetic patterns of traditional drugs. At present, research is being envisaged in the field of novel nano-pharmaceutical design, such as liposome, polymer NPs, bio-NPs, and inorganic NPs, so as to make chemotherapy effective and long-lasting. Till now, a number of studies have been conducted using a wide range of nanocarriers for the treatment of solid tumors including lung, breast, pancreas, brain, and liver. To provide a reference for the further application of chemodrug-loaded nanoformulations, this review gives an overview of the recent development of nanocarriers, and the updated status of their use in the treatment of several solid tumors.

S,S-Tetrazine-Based Hydrogels with Visible Light Cleavable Properties for On-Demand Anticancer Drug Delivery.

Photocleavable hydrogels are of great importance in the field of controlled drug delivery, stem cell fate regulation, surface patterning, and intelligent devices. However, the development of novel photocleavable gel systems by visible light is usually met with challenges such as the lack of efficient and tunable photocleavable groups and reactions. Herein, we reported the facile fabrication of a new type of photocleavable hydrogels by the direct gelation of 4-arm thiol-terminated polyethylene glycol with 3,6-dichloro-1,2,4,5-tetrazine via the formation of S,S-tetrazine linkages. The prepared hydrogels underwent efficient degradation upon irradiation by ultraviolet or green light, and the degradation kinetics could be significantly promoted by hydrogen peroxide. Correspondingly, the hydrogels loaded with calcium peroxide microparticles or glucose oxidase/catalase enzymes enabled the precise and efficient in vivo photocontrol of gel degradation and drug release for cancer treatment. This work offers a promising and facile strategy towards the fabrication of visible light cleavable hydrogels with tunable and on-demand drug release properties.

Potency and Selectivity of SMAC/DIABLO Mimetics in Solid Tumor Therapy.

Aiming to promote cancer cell apoptosis is a mainstream strategy of cancer therapy. The second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis protein (IAP)-binding protein with low pI (DIABLO) protein is an essential and endogenous antagonist of inhibitor of apoptosis proteins (IAPs). SMAC mimetics (SMs) are a series of synthetically chemical compounds. Via database analysis and literature searching, we summarize the potential mechanisms of endogenous SMAC inefficiency, degradation, mutation, releasing blockage, and depression. We review the development of SMs, as well as preclinical and clinical outcomes of SMs in solid tumor treatment, and we analyze their strengths, weaknesses, opportunities, and threats from our point of view. We also highlight several questions in need of further investigation.

[Development and application of optogenetic tools].

Dynamic variations of the cell microenvironment can affect cell differentiation, cell signaling pathways, individual growth, and disease. Optogenetics combines gene-encoded protein expression with optical controlling, and offers a novel, reversible, non-invasive and spatiotemporal-specific research tool to dynamically or reversibly regulate cell signaling pathways, subcellular localization and gene expression. This review summarizes the types of optogenetic components and the involved cellular signaling pathways, and explores the application and future prospects of the light-controlled cell signaling pathways.