Identification and culture of functional salivary gland ductal epithelial cells.

Sialadenitis is a prevalent salivary gland disease resulting in decreased salivary flow rate. To date, little is known about the exact changes and mechanism of ductal cells in sialadenitis. This study aims to establish an efficient method to identify and isolate ductal cells, thereby facilitating further research on this specific cell type. Immunofluorescence for cytokeratin 13 and cytokeratin 19 was conducted in salivary glands to confirm their specificity as ductal cell markers. The dissected ducts were assessed through PCR and Western blot of cytokeratin 19 and digested by dispase and collagenase. The functionality of the isolated ductal cells was determined by measuring intracellular calcium. Cytokeratin 19 and cytokeratin 13 were expressed in all segments of human ducts. Cytokeratin 19 was limited to ducts excluding granular convoluted tubules in rat and mouse. The purities of the obtained ductal cells were approximately 98% in humans and 93% in rats. Furthermore, intracellular free calcium increased with time and concentration of carbachol treatment. Cytokeratin 19 serves as a dependable marker for identifying ductal cells in salivary glands, except for granular convoluted tubules. Moreover, we have successfully developed an efficient method for isolating ductal cells from salivary glands.

The polyextremophile Natranaerobius thermophilus adopts a dual adaptive strategy to long-term salinity stress, simultaneously accumulating compatible solutes and K.

The bacterium Natranaerobius thermophilus is an extremely halophilic alkalithermophile that can thrive under conditions of high salinity (3.3-3.9 M Na+), alkaline pH (9.5), and elevated temperature (53°C). To understand the molecular mechanisms of salt adaptation in N. thermophilus, it is essential to investigate the protein, mRNA, and key metabolite levels on a molecular basis. Based on proteome profiling of N. thermophilus under 3.1, 3.7, and 4.3 M Na+ conditions compared to 2.5 M Na+ condition, we discovered that a hybrid strategy, combining the "compatible solute" and "salt-in" mechanisms, was utilized for osmotic adjustment dur ing the long-term salinity adaptation of N. thermophilus. The mRNA level of key proteins and the intracellular content of compatible solutes and K+ support this conclusion. Specifically, N. thermophilus employs the glycine betaine ABC transporters (Opu and ProU families), Na+/solute symporters (SSS family), and glutamate and proline synthesis pathways to adapt to high salinity. The intracellular content of compatible solutes, including glycine betaine, glutamate, and proline, increases with rising salinity levels in N. thermophilus. Additionally, the upregulation of Na+/ K+/ H+ transporters facilitates the maintenance of intracellular K+ concentration, ensuring cellular ion homeostasis under varying salinities. Furthermore, N. thermophilus exhibits cytoplasmic acidification in response to high Na+ concentrations. The median isoelectric points of the upregulated proteins decrease with increasing salinity. Amino acid metabolism, carbohydrate and energy metabolism, membrane transport, and bacterial chemotaxis activities contribute to the adaptability of N. thermophilus under high salt stress. This study provides new data that support further elucidating the complex adaptation mechanisms of N. thermophilus under multiple extremes.IMPORTANCEThis study represents the first report of simultaneous utilization of two salt adaptation mechanisms within the Clostridia class in response to long-term salinity stress.

Circulating extracellular vesicle-derived miR-1299 disrupts hepatic glucose homeostasis by targeting the STAT3/FAM3A axis in gestational diabetes mellitus.

BACKGROUND: Extracellular vesicles (EVs) are membrane-enclosed structures containing lipids, proteins, and RNAs that play a crucial role in cell-to-cell communication. However, the precise mechanism through which circulating EVs disrupt hepatic glucose homeostasis in gestational diabetes mellitus (GDM) remains unclear. RESULTS: Circulating EVs isolated from human plasma were co-cultured with mammalian liver cells to investigate the potential induction of hepatic insulin resistance by GDM-EVs using glucose output assays, Seahorse assays, metabolomics, fluxomics, qRT-PCR, bioinformatics analyses, and luciferase assays. Our findings demonstrated that hepatocytes exposed to GDM-EVs exhibited increased gluconeogenesis, attenuated energy metabolism, and upregulated oxidative stress. Particularly noteworthy was the discovery of miR-1299 as the predominant miRNA in GDM-EVs, which directly targeting the 3'-untranslated regions (UTR) of STAT3. Our experiments involving loss- and gain-of-function revealed that miR-1299 inhibits the insulin signaling pathway by regulating the STAT3/FAM3A axis, resulting in increased insulin resistance through the modulation of mitochondrial function and oxidative stress in hepatocytes. Moreover, experiments conducted in vivo on mice inoculated with GDM-EVs confirmed the development of glucose intolerance, insulin resistance, and downregulation of STAT3 and FAM3A. CONCLUSIONS: These results provide insights into the role of miR-1299 derived from circulating GDM-EVs in the progression of insulin resistance in hepatic cells via the STAT3/FAM3A axis and downstream metabolic reprogramming.

Development and validation of radiology-clinical statistical and machine learning model for stroke-associated pneumonia after first intracerebral haemorrhage.

BACKGROUND: Society is burdened with stroke-associated pneumonia (SAP) after intracerebral haemorrhage (ICH). Cerebral small vessel disease (CSVD) complicates clinical manifestations of stroke. In this study, we redefined the CSVD burden score and incorporated it into a novel radiological-clinical prediction model for SAP. MATERIALS AND METHODS: A total of 1278 patients admitted to a tertiary hospital between 1 January 2010 and 31 December 2019 were included. The participants were divided into training and testing groups using fivefold cross-validation method. Four models, two traditional statistical models (logistic regression and ISAN) and two machine learning models (random forest and support vector machine), were established and evaluated. The outcomes and baseline characteristics were compared between the SAP and non-SAP groups. RESULTS: Among the of 1278 patients, 281(22.0%) developed SAP after their first ICH. Multivariate analysis revealed that the logistic regression (LR) model was superior in predicting SAP in both the training and testing groups. Independent predictors of SAP after ICH included total CSVD burden score (OR, 1.29; 95% CI, 1.03-1.54), haematoma extension into ventricle (OR, 2.28; 95% CI, 1.87-3.31), haematoma with multilobar involvement (OR, 2.14; 95% CI, 1.44-3.18), transpharyngeal intubation operation (OR, 3.89; 95% CI, 2.7-5.62), admission NIHSS score ≥ 10 (OR, 2.06; 95% CI, 1.42-3.01), male sex (OR, 1.69; 95% CI, 1.16-2.52), and age ≥ 67 (OR, 2.24; 95% CI, 1.56-3.22). The patients in the SAP group had worse outcomes than those in the non-SAP group. CONCLUSION: This study established a clinically combined imaging model for predicting stroke-associated pneumonia and demonstrated superior performance compared with the existing ISAN model. Given the poor outcomes observed in patients with SAP, the use of individualised predictive nomograms is vital in clinical practice.

Engineering Escherichia coli for l-homoserine production.

l-homoserine, a nonprotein amino acid, is used to synthesize many active substances in the industry. Here, to develop a robust l-homoserine-producing strain, Escherichia coli W3110 was used as a chassis to be engineered. Based on a previous construct with blocked competing routes for l-homoserine synthesis, five genes were overexpressed by promoter replacement strategy to increase the l-homoserine production, including enhancement of precursors for l-homoserine synthesis (ppc, thrA, and asd), reinforcement of the NADPH supply (pntAB) and efflux transporters (rhtA) to improve the l-homoserine production. However, the plasmid losing was to blame for the wildly fluctuating fermentation performance of engineered strains, ranging between 2.1 and 6.2 g/L. Then, a hok/sok toxin/antitoxin system was introduced into the free plasmid expression cassette to maintain the genetic stability of the episomal plasmid; consequently, the plasmid-losing rate sharply decreased, resulting in the engineered strain SHL17, which exhibited excellent stability in l-homoserine production, with 6.3 g/L in shake flasks and 44.4 g/L in a 5-L fermenter without antibiotic addition. This work verified the effective use of the hok/sok toxin/antitoxin system combined with promoter engineering to improve the genetic stability of E. coli episomal plasmids without antibiotics.

Evaluation of the TRIP13 level in breast cancer and insights into potential molecular pathways.

TRIP13 is a member of the large superfamily of the AAA + ATPase proteins and is associated with a variety of activities. Emerging evidence has shown that TRIP13 may serve as an oncogene. However, the function of TRIP13 in breast cancer (BC) has not yet been elucidated. Here, a variety of bioinformatic tools and laboratory experiments were combined to analyse the expression patterns, prognostic value and functional network of TRIP13 in BC. Multiple databases and immunohistochemistry (IHC) indicated a higher TRIP13 expression in BC tissue compared with normal tissue. TRIP13 was highly expressed in lung metastatic lesions compared with primary tumours in a 4T1 cell implantation BALB/c mouse model of BC. Kaplan-Meier plots also revealed that high TRIP13 expression correlated with poor survival in patients with BC. Furthermore, gene set enrichment analysis revealed that TRIP13 was primarily enriched in the signalling pathway of PI3K-AKT-mTOR. Suppressing TRIP13 could inhibit the expression of related genes, as well as the proliferation and migration of BC cell. Finally, 10 hub genes with a high score of connectivity were filtered from the protein-protein interaction (PPI) network, including MAD2L1, CDC20, CDC5L, CDK1, CCNA2, BUB1B, RAD51, SPO11, KIF11 and AURKB. Thus, TRIP13 may be a promising prognostic biomarker and an effective therapeutic target for BC.

One-pot biosynthesis of 7ß-hydroxyandrost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered mycolicibacterium neoaurum.

BACKGROUND: 7ß-hydroxylated steroids (7ß-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7ß-OHSt is still a challenge due to the lack of cheap 7ß-hydroxy precursor and the difficulty in regio- and stereo-selectively hydroxylation at the inert C7 site of steroids in industry. The conversion of phytosterols by Mycolicibacterium species to the commercial precursor, androst-4-ene-3,17-dione (AD), is one of the basic ways to produce different steroids. This study presents a way to produce a basic 7ß-hydroxy precursor, 7ß-hydroxyandrost-4-ene-3,17-dione (7ß-OH-AD) in Mycolicibacterium, for 7ß-OHSt synthesis. RESULTS: A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7ß-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7ß-OH-AD was increased from 34.24 mg L- 1 to 66.25 mg L- 1. To further enhance the performance of 7ß-OH-AD producing strain, the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) for the activity of mP450-BM3-0 was optimized by introducing an NAD kinase (NADK) and a glucose-6-phosphate dehydrogenase (G6PDH). Finally, the engineered strain could produce 164.52 mg L- 1 7ß-OH-AD in the cofactor recycling and regeneration system. CONCLUSIONS: This was the first report on the one-pot biosynthesis of 7ß-OH-AD from the conversion of cheap phytosterols by an engineered microorganism, and the yield was significantly increased through the mutation of mP450-BM3 combined with overexpression of NADK and G6PDH. The present strategy may be developed as a basic industrial pathway for the commercial production of high value products from cheap raw materials.

Production of sesquiterpene patchoulol in mitochondrion-engineered Saccharomyces cerevisiae.

Patchoulol is a natural sesquiterpene, which is widely used in perfumes and cosmetics. In the work, the mitochondria of S. cerevisiae were engineered for patchoulol production. The patchoulol titer of mitochondria-compartmentalized strain (1.79 mg/L) was 2.71-fold higher than that of control strain (0.66 mg/L) using genome-integrated patchoulol synthase, indicating that mitochondria compartmentation resulted in higher concentration of FPP (farnesyl pyrophosphate) precursor for patchoulol production. Moreover, when fused FPP synthase and patchoulol synthase was overexpressed in the strain with a mitochondria-localized DMAPP (dimethylallyl diphosphate) pathway, the production of patchoulol increased significantly to 19.24 mg/L, indicating more precursors were provided for patchoulol production. Nevertheless, the introduction of excess foreign proteins into mitochondria might cause a certain stress on mitochondria and showed a negative effect on the growth of yeast cells, which could hinder the expression of foreign pathways and reduce the patchoulol production. In conclusion, mitochondria-engineered yeast cells showed important potential for the enhanced biosynthesis of patchoulol, and further engineering could be considered based on the present work.

Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction.

Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.

The yeast peroxisome: A dynamic storage depot and subcellular factory for squalene overproduction.

Engineering microbes to produce terpenes from renewable feedstock is a promising alternative to traditional production approaches. Generally, terpenes are not readily secreted by microbial cells, and their distribution within cells is usually obscure and often a restricting factor for the overproduction of terpenes due to the storage limitation. Here, we determined that squalene overproduced in the cytoplasm of Saccharomyces cerevisiae was distributed in a form similar to oil droplets. Interestingly, these suspected oil droplets were confirmed to be inflated peroxisomes that were swollen along with the production of squalene, indicating that peroxisomes in S. cerevisiae are dynamic depots for the storage of squalene. In view of this, harnessing peroxisomes as subcellular compartments for squalene synthesis was performed, achieving a 138-fold improvement in squalene titer (1312.82 mg/L) relative to the parent strain, suggesting that the peroxisome of S. cerevisiae is an efficient subcellular factory for the synthesis of terpenes. By dual modulation of cytoplasmic and peroxisomal engineering, the squalene titer was further improved to 1698.02 mg/L. After optimizing a two-stage fed-batch fermentation method, the squalene titer reached 11.00 g/L, the highest ever reported. This provides new insight into the synthesis and storage of squalene in peroxisomes and reveals the potential of harnessing peroxisomes to overproduce terpenes in S. cerevisiae through dual cytoplasmic-peroxisomal engineering.

Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae.

A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4 days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.

Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1N and KshA2N, were characterized and further engineered in a sterol-digesting strain, Mycobacterium neoaurum ATCC 25795, to construct androstenone-producing strains. kshA1N is a member of the gene cluster encoding sterol catabolism enzymes, and its transcription exhibited a 4.7-fold increase under cholesterol induction. Furthermore, null mutation of kshA1N led to the stable accumulation of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined kshA2N to be a redundant form of kshA1N Through a combined modification of kshA1N, kshA2N, and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g liter-1 ADD from the transformation of 20 g liter-1 phytosterols in 168 h. Moreover, we improved a previously established 9α-hydroxy-AD-producing strain via the overexpression of a mutant KshA1N that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g liter-1 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from the transformation of 20.0 g liter-1 phytosterol in 120 h.IMPORTANCE Steroidal drugs are widely used for anti-inflammation, anti-tumor action, endocrine regulation, and fertility management, among other uses. The two main starting materials for the industrial synthesis of steroid drugs are phytosterol and diosgenin. The phytosterol processing is carried out by microbial transformation, which is thought to be superior to the diosgenin processing by chemical conversions, given its simple and environmentally friendly process. However, diosgenin has long been used as the primary starting material instead of phytosterol. This is in response to challenges in developing efficient microbial strains for industrial phytosterol transformation, which stem from complex metabolic processes that feature many currently unclear details. In this study, we identified two oxygenase homologues of 3-ketosteroid-9α-hydroxylase, KshA1N and KshA2N, in M. neoaurum and demonstrated their crucial role in determining the yield and variety of products from phytosterol transformation. This work has practical value in developing industrial strains for phytosterol biotransformation.

Establishment of a low-dosage-IPTG inducible expression system construction method in Escherichia coli.

The lac operon is a delicate inducible gene expression element in bacteria. To efficiently induce gene expression, a sufficient dosage of an inducer, usually that of 500-1000 µM isopropyl ß-D-1-thiogalactopyranoside (IPTG), is required to keep repressor LacI from its binding sites, which is a heavy cost burden in low-value-added products. So we propose a strategy to reduce the required dosage of IPTG by restricting LacI expression. To test this strategy, we employed a reconstructed IPTG inducible expression system based on lac operon, Promoter(lacO)-target gene-PtacL-lacI, where a modified promoter, Ptac, with a random synthetic library (PtacL) to instead of PlacI to optimize LacI expression in Escherichia coli. Finally, the PtacL mutant, PtacL4, which could maintain the same repression effect as the original PlacI while reducing the required dosage of IPTG from 500 to 20 µM, was selected. This method is simple and efficient and can be of a good reference point for attempts to reduce inducer concentration in the IPTG or similar inducible expression systems.

New compounds from a hydrothermal vent crab-associated fungus Aspergillus versicolor XZ-4.

Three new quinazoline derivatives (1-3), one new oxepin-containing natural product (4) and four new cyclopenin derivatives (5-7 and 9) have been isolated from an EtOAc extract of the Taiwan Kueishantao hydrothermal vent crab-associated fungus Aspergillus versicolor XZ-4. Their planar structures were established by HRMS, 1D and 2D NMR spectroscopic data analyses. The absolute configurations for compounds 1 and 4 were determined by chiral phase HPLC analysis of their hydrolysis products. The absolute configurations of 2, 3 and 7 were defined mainly by comparison of the quantum chemical TDDFT calculated and the experimental ECD spectra, and the absolute configuration of 5 was deduced from comparison of the optical rotation values reported in the literature. The presence of two atropisomers of 5 was established by NOE analyses. The Ile & Val units in compounds 1-3 allowed the assignment of a new quinazoline skeleton and it's the first time the configuration of isoleucine in the quinazoline skeleton was defined. A series of 7-methoxy cyclopenin derivatives were reported for the first time in this study. The bioevaluation of compounds 5, 7, 8 and 9 revealed inhibitory activities against E. coli at MIC values around 32 µg mL-1.

Cell-penetrating and endoplasmic reticulum-locating TAT-IL-24-KDEL fusion protein induces tumor apoptosis.

Interleukin-24 (IL-24) is a unique IL-10 family cytokine that could selectively induce apoptosis in cancer cells without harming normal cells. Previous research demonstrated that intracellular IL-24 protein induces an endoplasmic reticulum (ER) stress response only in cancer cells, culminating in apoptosis. In this study, we developed a novel recombinant fusion protein to penetrate into cancer cells and locate on ER. It is composed of three distinct functional domains, IL-24, and the targeting domain of transactivator of transcription (TAT) and an ER retention four-peptide sequence KDEL (Lys-Asp-Glu-Leu) that link at its NH2 and COOH terminal, respectively. The in vitro results indicated that TAT-IL-24-KDEL inhibited growth in bladder cancer cells, as well as in non-small cell lung cancer cell line and breast cancer cell line, but the normal human lung fibroblast cell line was not affected, indicating the cancer specificity of TAT-IL-24-KDEL. Western blot analysis showed that apoptosis activation was induced by TAT-IL-24-KDEL through the ER stress-mediated cell death pathway. Treatment with TAT-IL-24-KDEL significantly inhibited the growth of human H460 xenografts in nude mice, and the tumor growth inhibition was correlated with increased hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. These findings suggest that the artificially designed recombinant fusion protein TAT-IL-24-KDEL may be highly effective in cancer therapy and worthy of further evaluation and development.

RNA-Seq analysis uncovers non-coding small RNA system of Mycobacterium neoaurum in the metabolism of sterols to accumulate steroid intermediates.

BACKGROUND: Understanding the metabolic mechanism of sterols to produce valuable steroid intermediates in mycobacterium by a noncoding small RNA (sRNA) view is still limited. In the work, RNA-seq was implemented to investigate the noncoding transcriptome of Mycobacterium neoaurum (Mn) in the transformation process of sterols to valuable steroid intermediates, including 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA). RESULTS: A total of 263 sRNA candidates were predicted from the intergenic regions in Mn. Differential expression of sRNA candidates was explored in the wide type Mn with vs without sterol addition, and the steroid intermediate producing Mn strains vs wide type Mn with sterol addition, respectively. Generally, sRNA candidates were differentially expressed in various strains, but there were still some shared candidates with outstandingly upregulated or downregulated expression in these steroid producing strains. Accordingly, four regulatory networks were constructed to reveal the direct and/or indirect interactions between sRNA candidates and their target genes in four groups, including wide type Mn with vs without sterol addition, 9OHAD, ADD, and BNA producing strains vs wide type Mn with sterol addition, respectively. Based on these constructed networks, several highly focused sRNA candidates were discovered to be prevalent in the networks, which showed comprehensive regulatory roles in various cellular processes, including lipid transport and metabolism, amino acid transport and metabolism, signal transduction, cell envelope biosynthesis and ATP synthesis. To explore the functional role of sRNA candidates in Mn cells, we manipulated the overexpression of candidates 131 and 138 in strain Mn-9OHAD, which led to enhanced production of 9OHAD from 1.5- to 2.3-fold during 6 d' fermentation and a slight effect on growth rate. CONCLUSIONS: This study revealed the complex and important regulatory roles of noncoding small RNAs in the metabolism of sterols to produce steroid intermediates in Mn, further analysis of which will promote the better understanding about the molecular metabolism of these sRNA candidates and open a broad range of opportunities in the field.

Single nucleotide polymorphism analysis for the production of valuable steroid intermediates in Mycobacterium neoaurum.

OBJECTIVES: To investigate single nucleotide polymorphism (SNP) in the transformation process of phytosterol to valuable steroid intermediates in three steroid-producing Mycobacterium neoaurum strains using deep sequencing and bioinformation analysis. RESULTS: The assembled contig sequences from RNA sequencing of strains producing 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA) were analyzed for the presence of putative SNPs for steroid catabolism. 413, 375, and 491 SNPs were detected in the coding domain sequences and non-coding domain sequences of RNA sequencing reads of M. neoaurum strains producing 9OHAD, ADD, and BNA, respectively. Special attention was focused on SNPs associated with genes showing differential expression at proteome level, including the genes for sterol catabolism, glycerol catabolic process, signal transduction systems, transport system and energy metabolism. CONCLUSIONS: The work facilitates the understanding of underlying genetic changes that may be responsible for steroid accumulation in M. neoaurum and is useful for its targeted genetic engineering.

An Unusual Conformational Isomer of Verrucosidin Backbone from a Hydrothermal Vent Fungus, Penicillium sp. Y-50-10.

A new verrucosidin derivative, methyl isoverrucosidinol (1), was isolated from the marine fungus Penicillium sp. Y-50-10, dwelling in sulfur rich sediment in the Kueishantao hydrothermal vents off Taiwan. The structure was established by spectroscopic means including HRMS and 2D-NMR spectroscopic analysis. The absolute configuration was defined mainly by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Among hitherto known compounds with a verrucosidine backbone isolated from natural resource, compound 1 represents the first example of a new conformational isomer of its skeleton, exhibiting antibiotic activity against Bacillus subtilis with MIC value 32 µg/mL.

Soluble expression, rapid purification, and characterization of human interleukin-24 (IL-24) using a MBP-SUMO dual fusion system in Escherichia coli.

Interleukin-24 (IL-24), a cytokine belonging to the IL-10 family, can selectively induce apoptosis in a broad range of tumor cells without harming normal cells. The efficient and soluble expression of bioactive recombinant IL-24 in Escherichia coli remains an obstacle because of aggregation and insufficient yield. In this study, a fusion of the small ubiquitin-related modifier (SUMO) or maltose-binding protein (MBP) has shown potential in facilitating the produce of IL-24. Thus, a new construct for MBP-SUMO-IL-24 expression would be a promising approach. Our results showed that the MBP-SUMO-IL-24 fusion protein was efficiently expressed as a soluble protein. SUMO protease-mediated cleavage at the SUMO/IL-24 junction released the recombinant IL-24 from the fusion protein. In addition, a His6 tag fused upstream of SUMO allowed for one-step purification through nickel affinity chromatography. Cleavage of the MBP-SUMO tag on the column resulted in the release of purified IL-24 and simplified the purification process. The final yield of IL-24 with approximately 90 % purity was 19 mg/L in flask fermentation. In vitro activity assays demonstrated that the purified IL-24 could induce apoptosis in MCF-7 breast cancer cells, but not normal NHLF cells, in a dose-dependent manner. In summary, we developed a novel method to express soluble and bioactive IL-24 protein in prokaryotic cells.

TAT-RhoGDI2, a novel tumor metastasis suppressor fusion protein: expression, purification and functional evaluation.

Rho GDP dissociation inhibitor 2 (RhoGDI2) was identified as a functional metastasis suppressor in human bladder cancer, suggesting that increasing the RhoGDI2 level may represent a promising therapeutic strategy. It has been shown that the transactivator of transcription (TAT) protein from HIV-1 is able to efficiently deliver various biological molecules into several cell types. In this study, TAT peptide was fused with the N-terminus of RhoGDI2, and the resulting TAT-RhoGDI2 fragment was inserted into the pGEX-6p-1 plasmid and expressed as a glutathione S-transferase (GST)/TAT-RhoGDI2 fusion protein in Escherichia coli BL21(DE3) cells. A two-step purification strategy involving glutathione sepharose chromatography and PreScission protease cleavage was developed to purify TAT-RhoGDI2; subsequently, the identification of the involved macromolecules was achieved by Western blot. The final product, TAT-RhoGDI2, was obtained at a concentration of 112 mg/L. This is the first report on the efficient production of bioactive TAT-RhoGDI2 through a gene-engineering approach in E. coli. Using flow cytometry, we found that the TAT-RhoGDI2 fusion proteins could penetrate into bladder cancer cells with an extremely high efficiency. In vitro scratch and transwell assay and the migration/invasion behavior of UMUC3 cells were strongly reduced by the treatment with TAT-RhoGDI2. These studies support the use of the TAT-RhoGDI2 protein in tumor metastasis therapy.