Evaluation of cardiac index and right ventricular hypertrophy index in rats under a chronic hypoxic environment at high altitude.

High-altitude areas are characterized by low pressure and hypoxia, which have a significant impact on various body systems. This study aimed to investigate the alterations in cardiac index and right ventricular hypertrophy index(RVHI) in rats at different altitudes.Twenty-one male Sprague-Dawley (SD) rats aged 4 weeks were randomly divided into three groups based on altitude. The rats were raised for 28 weeks and then transferred to Qinghai University Plateau Medicine Laboratory. Body weight was measured, heart organs were isolated and weighed, and cardiac index and right ventricular hypertrophy index were determined. Statistical analysis was performed on the data from the three groups. Compared with the plain group, the body weight of the middle-altitude group was significantly decreased (P < 0.05), and cardiac index, RVHI-1, RVHI-2 increased significantly ((P < 0.05). The body weight, whole heart mass, right ventricular mass were significantly decreased in high-altitude group (P < 0.05), RVHI-1 and RVHI-2 were significantly increased (P < 0.05). Compared with the middle-altitude group, the body weight, whole heart mass and right ventricular mass of the high-altitude group were significantly decreased (P < 0.05), and RVHI-1 and RVHI-2 were significantly increased (P < 0.05). Increasing altitude led to a decrease in body weight, whole heart mass, and right ventricular mass in rats, indicating structural changes in the right heart. Additionally, the proportion of right heart to body weight and whole heart increased with altitude.

Evaluation of Myocardial Microcirculation in Rats under a High-Altitude Hypoxic Environment by Computed Tomography Myocardial Perfusion Imaging.

This study aims to examine the changes in myocardial microcirculation in rats in a high-altitude hypoxic environment via computed tomography (CT) myocardial perfusion imaging technology. Rats in two groups were raised in different environments from 4 weeks of age for a period of 24 weeks. At 28 weeks of age, both groups underwent CT myocardial perfusion scanning, and the following myocardial perfusion parameters were measured: time to peak (TTP), mean transit time (MTT), blood flow (BF), and blood volume (BV). Following the scan, the rats were sacrificed, the cardiac index and right ventricular hypertrophy index were obtained, and hematoxylin-eosin (HE) staining was utilized to observe the pathological changes in the myocardium. In the group of rats that are subject to a high-altitude hypoxic environment for 24 weeks (the high-altitude group), the TTP and MTT values were increased (P < 0.05), the BF and BV values were lower (P < 0.05), the right heart mass was higher (P < 0.05) than that in the low-altitude group. As shown by the pathological results of HE staining, the gap between cardiomyocytes in the high-altitude group was widened, the arrangement of cardiomyocytes was irregular, and the cells were filled with a few fat vacuoles. The myocardial microcirculation is altered in a high-altitude hypoxic environment. In particular, the myocardium is in a state of inadequate perfusion, the BF in the myocardium slows down, and the right heart displays compensatory hypertrophy.

O-methyltransferase-like enzyme catalyzed diazo installation in polyketide biosynthesis.

Diazo compounds are rare natural products possessing various biological activities. Kinamycin and lomaiviticin, two diazo natural products featured by the diazobenzofluorene core, exhibit exceptional potency as chemotherapeutic agents. Despite the extensive studies on their biosynthetic gene clusters and the assembly of their polyketide scaffolds, the formation of the characteristic diazo group remains elusive. L-Glutamylhydrazine was recently shown to be the hydrazine donor in kinamycin biosynthesis, however, the mechanism for the installation of the hydrazine group onto the kinamycin scaffold is still unclear. Here we describe an O-methyltransferase-like protein, AlpH, which is responsible for the hydrazine incorporation in kinamycin biosynthesis. AlpH catalyses a unique SAM-independent coupling of L-glutamylhydrazine and polyketide intermediate via a rare Mannich reaction in polyketide biosynthesis. Our discovery expands the catalytic diversity of O-methyltransferase-like enzymes and lays a strong foundation for the discovery and development of novel diazo natural products through genome mining and synthetic biology.

Unexpected Role of a Short-Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis.

We investigated the biosynthetic pathway of type II polyketide murayaquinone. The murayaquinone biosynthetic cluster contains genes for three putative short-chain dehydrogenase/reductase family enzymes including MrqF and MrqH with known functions and MrqM with unclear function. We report the functional characterization of MrqM for its role in murayaquinone biosynthesis. Our gene deletion experiment and structural elucidation of the accumulated intermediates revealed that MrqM is related with the second polyketide ring cyclization, because the inactivation of mrqM resulted in the accumulation of an off-pathway intermediate SEK43 and disrupted the second and third ring cyclization. Site-directed mutagenesis studies showed that two conserved residues in MrqM and homologous proteins Y151 and K155 are essential for its activity. The previously proposed second/third ring cyclase, MrqD, might instead play another important role in the chain releasing step of the murayaquinone biosynthesis.

A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S.

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of ß-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the ß-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron-sulfur cluster that is necessary for activity. These findings provide an example of an iron-sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)-dependent amide bond formation in a natural product biosynthetic pathway.

Challenges of functional expression of complex polyketide biosynthetic gene clusters.

Polyketide natural products are valuable sources of bioactive molecules such as nutraceuticals and pharmaceuticals. The tremendous development of the genome sequence database revealed that the majority of the biosynthetic gene clusters (BGCs) are cryptic. Activation of these cryptic BGCs and identification of the related products is essential for finding more lead compounds for pharmaceuticals. On the other hand, 99% of microbes in nature cannot be cultured in regular conditions, which greatly hinders the efforts to explore their biosynthetic potentials. Expression of polyketide BGCs in heterologous hosts with better growth, good genetic characteristics, and amenable molecular tools is a robust approach to identify new polyketides and characterize their biosynthesis. This review outlines the challenges in the heterologous production of polyketide BGCs of bacterial origins.

Offloading Role of a Discrete Thioesterase in Type II Polyketide Biosynthesis.

Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KSα), chain length factor (KSß), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KSα or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

Isolation of Thermostable Lignocellulosic Bacteria From Chicken Manure Compost and a M42 Family Endocellulase Cloning From Geobacillus thermodenitrificans Y7.

The composting ecosystem provides a potential resource for finding new microorganisms with the capability for cellulose degradation. In the present study, Congo red method was used for the isolating of thermostable lignocellulose-degrading bacteria from chicken manure compost. A thermophilic strain named as Geobacillus thermodenitrificans Y7 with acid-resident property was successfully isolated and employed to degrade raw switchgrass at 60°C for 5 days, which resulted in the final degradation rates of cellulose, xylan, and acid-insoluble lignin as 18.64, 12.96, and 17.21%, respectively. In addition, GC-MS analysis about aromatic degradation affirm the degradation of lignin by G. thermodenitrificans Y7. Moreover, an endocellulase gene belong to M42 family was successfully cloned from G. thermodenitrificans Y7 and expressed in Escherichia coli BL21. Recombinant enzyme Cel-9 was purified by Ni-NTA column based the His-tag, and the molecular weight determined as 40.4 kDa by SDA-PAGE. The characterization of the enzyme Cel-9 indicated that the maximum enzyme activity was realized at 50°C and pH 8.6 and, Mn2+ could greatly improve the CMCase enzyme activity of Cel-9 at 10 mM, which was followed by Fe2+ and Co2+. Besides, it also found that the ß-1,3-1,4, ß-1,3, ß-1,4, and ß-1,6 glucan linkages all could be hydrolyzed by enzyme Cel-9. Finally, during the application of enzyme Cel-9 to switchgrass, the saccharification rates achieved to 1.81 ± 0.04% and 2.65 ± 0.03% for 50 and 100% crude enzyme, respectively. All these results indicated that both the strain G. thermodenitrificans Y7 and the recombinant endocellulase Cel-9 have the potential to be applied to the biomass industry.