Chloroplast genome analysis and evolutionary insights in the versatile medicinal plant Calendula officinalis L.

Calendula officinalis L.is a versatile medicinal plant with numerous applications in various fields. However, its chloroplast genome structure, features, phylogeny, and patterns of evolution and mutation remain largely unexplored. This study examines the chloroplast genome, phylogeny, codon usage bias, and divergence time of C. officinalis, enhancing our understanding of its evolution and adaptation. The chloroplast genome of C. officinalis is a 150,465 bp circular molecule with a G + C content of 37.75% and comprises 131 genes. Phylogenetic analysis revealed a close relationship between C. officinalis, C. arvensis, and Osteospermum ecklonis. A key finding is the similarity in codon usage bias among these species, which, coupled with the divergence time analysis, supports their close phylogenetic proximity. This similarity in codon preference and divergence times underscores a parallel evolutionary adaptation journey for these species, highlighting the intricate interplay between genetic evolution and environmental adaptation in the Asteraceae family. Moreover unique evolutionary features in C. officinalis, possibly associated with certain genes were identified, laying a foundation for future research into the genetic diversity and medicinal value of C. officinalis.

New insights into the relationship between the average nucleotide identity and the digital DNA-DNA hybridization values in the genus Amycolatopsis and Amycolatopsis cynarae sp. nov., a novel actinobacterium from the rhizosphere soil of Cynara scolymus, and proposal of Amycolatopsis niigatensis as a synonym of Amycolatopsis echigonensis based on comparative genomic analysis.

At present, it is widely believed that a 95-96% average nucleotide identity (ANI) value is equivalent to a 70% digital DNA-DNA hybridization (dDDH) value in the prokaryotic taxonomy. However, in the present study, comparative genome analysis of 29 pairs of Amycolatopsis type strains revealed that a 70% dDDH value did not correspond to a 95-96% ANI based on the MuMmer ultra-rapid aligning tool (ANIm) but approximately corresponded to a 96.6% ANIm value in the genus Amycolatopsis. Based on this corresponding relationship, phenotypic and chemotaxonomical characteristics, as well as phylogenetic analysis, an actinobacterial strain HUAS 11-8T isolated from the rhizosphere soil of Cynara scolymus, was subjected to a polyphasic taxonomic characterization. Based on EzBioCloud alignment, it was found that strain HUAS11-8T had the 16S rRNA gene similarities of 99.78% with A. rhizosphaerae JCM 32589T, 97.8% with A. dongchuanensis YIM 75904T, and < 97.8% sequence similarities to other Amycolatopsis species. Phylogenetic analysis of 16S rRNA gene sequences and whole-genome sequences revealed that strain HUAS 11-8T was closely related to A. rhizosphaerae JCM 32589T. ANIm and dDDH values between strains HUAS 11-8T and A. rhizosphaerae JCM 32589T were 96.3 and 68.5%, respectively, lower than the 96.6 and 70% thresholds recommended for the delineation of a novel Amycolatopsis species. Consequently, strain HUAS 11-8T should represent a novel Amycolatopsis species, for which the name Amycolatopsis cynarae sp. nov. (type strain HUAS 11-8T = MCCC 1K08337T = JCM 35980T) is proposed. Furthermore, based on comparative genomic analysis and rule 42 of the Prokaryotic Code, we propose that Amycolatopsis niigatensis is a later heterotypic synonym of Amycolatopsis echigonensis.

Artichoke (Cynara scolymus L.) water extract alleviates palmitate-induced insulin resistance in HepG2 hepatocytes via the activation of IRS1/PI3K/AKT/FoxO1 and GSK-3ß signaling pathway.

BACKGROUND: Artichoke (Cynara scolymus L.) is a typical element of a traditional Mediterranean diet and has potential health advantages for insulin resistance (IR) and type 2 diabetes mellitus (T2DM). This study aims to evaluate the effect and underlying mechanism of artichoke water extract (AWE) on palmitate (PA)-induced IR in human hepatocellular carcinoma (HepG2) cells. METHODS: The effect of AWE on cell viability was determined using CCK8 assay. Cellular glucose uptake, glucose consumption, glucose production, and glycogen content were assessed after AWE treatment. The gene expression and protein levels were examined by real-time polymerase chain reaction (qRT-PCR) and western blotting. RESULTS: The results showed that AWE dose-dependently increased cell viability in IR HepG2 cells (P < 0.01). AWE treatment significantly promoted glucose uptake and consumption, decreased glucose production, and increased the cellular glycogen content in IR HepG2 cells (P < 0.01). Mechanistically, AWE elevated the phosphorylation and total protein levels of major insulin signaling molecules in IR HepG2 cells, which resulted in a decrease in the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and the inhibition of glycogen synthase (GS) phosphorylation in IR HepG2 cells. Furthermore, the protective effect of AWE on IR HepG2 cells might be ascribed to the inhibition of the endoplasmic reticulum (ER) stress. CONCLUSION: We conclude that AWE may improve glucose metabolism by regulating IRS1/PI3K/AKT/FoxO1 and GSK-3ß signaling associated with the inhibition of ER stress in IR HepG2 cells induced by PA.

Streptomyces cynarae sp. nov., a novel actinomycete isolated from the leaves of Cynara scolymus L.

Strain HUAS 13-4T, a novel endophytic actinobacterium, was isolated from the leaves of Cynara scolymus L. collected from Changde City in China and characterized using a polyphasic approach. Based on 16S rRNA gene sequence analysis, strain HUAS 13-4T shared the highest sequence similarities to Streptomyces leeuwenhoekii C34T (98.90%), Streptomyces harenosi PRKS01-65T (98.83%) and Streptomyces glomeratus LMG 19903T (98.76%). Phylogenetic analysis of 16S rRNA gene sequence indicated that strain HUAS 13-4T was clustered together with Streptomyces bluensis ISP 5564T and Streptomyces cavernae SYSU K10008T. Phylogenomic analysis revealed that strain HUAS 13-4T was most closely related to S. glomeratus JCM 9091T. However, the average nucleotide identity and the digital DNA-DNA hybridization values between them were less than 96.7% and 70% cut-off points recommended for delineating species. Based on a comprehensive comparison of the genome sequences and phenotypic characteristics between strain HUAS 13-4T and its relative, strain HUAS 13-4T (= MCCC 1K08364T = JCM 35919T) should evidently represent a novel Streptomyces species, and the name Streptomyces cynarae sp. nov. is proposed.

Streptomyces argyrophyllae sp. nov., isolated from the rhizosphere soil of Cathaya argyrophylla.

Strain Jing01T, a novel actinomycete from rhizosphere soil of Cathaya argyrophylla, was identified using a polyphasic approach. 16S rRNA gene sequence analysis of strain Jing01T revealed that it was a member of the genus Streptomyces and shared 99.03%, 99.03%, 98.96%, 98.89%, 98.83%, 98.82%, 98.76%, 98.74%, 98.73%, 98.69% and 98.68% similarities to Streptomyces rochei NRRL B-2410T, Streptomyces naganishii NBRC 12892T, Streptomyces rubradiris JCM 4955T, Streptomyces anandii NRRL B-3590T, Streptomyces aurantiogriseus NBRC 12842T, Streptomyces mutabilis NBRC 12800T, Streptomyces rameus LMG 20326T, Streptomyces djakartensis NBRC 15409T, Streptomyces bangladeshensis JCM 14924T, Streptomyces andamanensis KCTC 29502T and Streptomyces tuirus NBRC 15617T, respectively. In phylogenetic trees constructed based on 16S rRNA gene sequences, strain Jing01T generated a separate branch at the middle of the clade, suggesting it could be a potential novel species. In phylogenomic tree, strain Jing01T was related to S. rubradiris JCM 4955T. In phylogenetic trees based on the gene sequences of atpD, gyrB, recA, rpoB and trpB, strain Jing01T was related to S. bangladeshensis JCM 14924T and S. rubradiris JCM 4955T. Whereas, the multilocus sequence analysis distance, average nucleotide identity and DNA-DNA hybridization values between them were much less than the species-level thresholds. This conclusion was further supported by phenotypic and chemotaxonomic analysis. Consequently, strain Jing01T represents a new Streptomyces species, for which the proposed name is Streptomyces argyrophyllae sp. nov. The type strain is Jing01T (= MCCC 1K05707T = JCM 35923T).

The diversity and abundance of bacterial and fungal communities in the rhizosphere of Cathaya argyrophylla are affected by soil physicochemical properties.

Cathaya argyrophylla is an ancient Pinaceae species endemic to China that is listed on the IUCN Red List. Although C. argyrophylla is an ectomycorrhizal plant, the relationship between its rhizospheric soil microbial community and soil properties related to the natural habitat remains unknown. High-throughput sequencing of bacterial 16S rRNA genes and fungal ITS region sequences was used to survey the C. argyrophylla soil community at four natural spatially distributed points in Hunan Province, China, and functional profiles were predicted using PICRUSt2 and FUNGuild. The dominant bacterial phyla included Proteobacteria, Acidobacteria, Actinobacteria, and Chloroflexi, and the dominant genus was Acidothermus. The dominant fungal phyla were Basidiomycota and Ascomycota, while Russula was the dominant genus. Soil properties were the main factors leading to changes in rhizosphere soil bacterial and fungal communities, with nitrogen being the main driver of changes in soil microbial communities. The metabolic capacities of the microbial communities were predicted to identify differences in their functional profiles, including amino acid transport and metabolism, energy production and conversion, and the presence of fungi, including saprotrophs and symbiotrophs. These findings illuminate the soil microbial ecology of C. argyrophylla, and provide a scientific basis for screening rhizosphere microorganisms that are suitable for vegetation restoration and reconstruction for this important threatened species.

Generation of attosecond micro bunched beam using ionization injection in laser wakefield acceleration.

Micro bunched electron beams with periodic longitudinal density modulation at optical wavelengths give rise to coherent light emission. In this paper, we show attosecond micro bunched beam generation and acceleration in laser-plasma wakefield via particle-in-cell simulations. Due to the near-threshold ionization with the drive laser, the electrons with phase-dependent distributions are non-linearly mapped to discrete final phase spaces. Electrons can preserve this initial bunching structure during the acceleration, leading to an attosecond electron bunch train after leaving the plasma with separations of the same time scale. The modulation of the comb-like current density profile is about 2k0 ∼ 3k0, where k0 is the wavenumber of the laser pulse. Such pre-bunched electrons with low relative energy spread may have potential in applications related to future coherent light sources driven by laser-plasma accelerators and broad application prospects in attosecond science and ultrafast dynamical detection.

Multivariate modular metabolic engineering for enhanced L-methionine biosynthesis in Escherichia coli.

BACKGROUND: L-Methionine is the only bulk amino acid that has not been industrially produced by the fermentation method. Due to highly complex and strictly regulated biosynthesis, the development of microbial strains for high-level L-methionine production has remained challenging in recent years. RESULTS: By strengthening the L-methionine terminal synthetic module via site-directed mutation of L-homoserine O-succinyltransferase (MetA) and overexpression of metAfbr, metC, and yjeH, L-methionine production was increased to 1.93 g/L in shake flask fermentation. Deletion of the pykA and pykF genes further improved L-methionine production to 2.51 g/L in shake flask fermentation. Computer simulation and auxotrophic experiments verified that during the synthesis of L-methionine, equimolar amounts of L-isoleucine were accumulated via the elimination reaction of cystathionine γ-synthetase MetB due to the insufficient supply of L-cysteine. To increase the supply of L-cysteine, the L-cysteine synthetic module was strengthened by overexpression of cysEfbr, serAfbr, and cysDN, which further increased the production of L-methionine by 52.9% and significantly reduced the accumulation of the byproduct L-isoleucine by 29.1%. After optimizing the addition of ammonium thiosulfate, the final metabolically engineered strain MET17 produced 21.28 g/L L-methionine in 64 h with glucose as the carbon source in a 5 L fermenter, representing the highest L-methionine titer reported to date. CONCLUSIONS: In this study, a high-efficiency strain for L-methionine production was derived from wild-type Escherichia coli W3110 by rational metabolic engineering strategies, providing an efficient platform for the industrial production of L-methionine.

Adaptive mechanisms of Bacillus to near space extreme environments.

Earth's near space is an extreme atmosphere environment with high levels of radiation, low atmospheric pressure and dramatic temperature fluctuations. The region is above the flight altitude of aircraft but below the orbit of satellites, which has special and Mars-like conditions for investigating the survival and evolution of life. Technical limitations including flight devices, payloads and technologies/methodologies hinder microbiological research in near space. In this study, we investigated microbial survival and adaptive strategies in near space using a scientific balloon fight mission and multi-omics analyses. Methods for sample preparation, storage, protector and vessel were optimized to prepare the exposed microbial samples. After 3 h 17 min of exposure at a float altitude of ~32 km, only Bacillus strains were alive with survival efficiencies of 0-10-6. Diverse mutants with significantly altered metabolites were generated, firstly proving that Earth's near space could be used as a new powerful microbial breeding platform. Multi-omics analyses of mutants revealed cascade changes at the genome, transcriptome and proteome levels. In response to environmental stresses, two mutants had similar proteome changes caused by different genomic mutations and mRNA expression levels. Metabolic network analysis combined with proteins' expression levels revealed that metabolic fluxes of EMP, PPP and purine synthesis-related pathways were significantly altered to increase/decrease inosine production. Further analysis showed that proteins related to translation, molecular chaperones, cell wall/membrane, sporulation, DNA replication/repair and anti-oxidation were significantly upregulated, enabling cells to efficiently repair DNA/protein damages and improve viability against environmental stress. Overall, these results revealed genetic and metabolic responses of Bacillus to the harsh conditions in near space, providing a research basis for bacterial adaptive mechanisms in extreme environments.

Water extract from artichoke ameliorates high-fat diet-induced non-alcoholic fatty liver disease in rats.

BACKGROUND: The "multiple-hit" hypothesis is currently the most widely accepted theory for non-alcoholic fatty liver disease (NAFLD) pathogenesis. The present study aimed to investigate the effects of the water extract of artichoke (WEA) on NAFLD and its underlying mechanism. METHODS: Rats were fed a high-fat diet (HFD) for 8 weeks to induce NAFLD and then treated with WEA at three doses (0.4, 0.8, and 1.6 g/kg body weight, BW) for 8 weeks. At the end of the intervention, serum biochemical parameters, hepatic antioxidant capacity, hepatic levels of pro-inflammatory cytokines, liver histopathology, hepatic inflammatory gene and lipid metabolism gene expression, and Akt and p-Akt (S473) protein levels were determined. RESULTS: The body weight, liver weight, liver triglyceride (TG) and serum levels of TG, total cholesterol, low-density lipoprotein cholesterol, alanine aminotransferase, aspartate aminotransferase, glucose, and insulin were all significantly reduced in the WEA-treated groups (0.8 and 1.6 g/kg BW) compared with the HFD group (P < 0.01). A significant decrease in hepatic content of malondialdehyde (P < 0.01) and glutathione (P < 0.01), as well as a significant increase in liver superoxide dismutase activity (P < 0.01) were observed in WEA-treated groups (0.8 and 1.6 g/kg BW) compared to the HFD group. In addition, there was a marked decrease in the hepatic levels of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in the WEA-treated groups compared to the HFD group (P < 0.01). In line with these findings, the histopathology of the livers of rats treated with WEA (0.8 and 1.6 g/kg BW) showed a decrease in steatosis, ballooning, and lobular inflammation. Mechanistically, the reduced hepatic TG content might be related to the downregulation of lipogenic genes (SREBP1c, FASN, SCD1) and upregulation of lipolytic gene (PPARα), and the improved insulin signaling might be associated with the observed increase in antioxidant activity and reduction in inflammation in the WEA-treated groups. CONCLUSION: The hepatoprotective role of WEA in NAFLD may be attributed to its anti-steatotic, antioxidant, anti-inflammatory, and anti-insulin resistance effects.

Differences in the Chloroplast Genome and Its Regulatory Network among Cathaya argyrophylla Populations from Different Locations in China.

Cathaya argyrophylla Chun et Kuang is a severely endangered, tertiary relict plant unique to China whose high physiological sensitivity to the environment, including photosensitivity, is likely closely related to its endangered status; however, the exact mechanism responsible has remained unknown due to the rarity of the plant and the difficulties involved in performing physiological studies on the molecular level. In this study, the chloroplast genomes of six C. argyrophylla populations sampled from different locations in China were characterized and compared. In addition, a gene regulatory network of the polymorphic chloroplast genes responsible for regulating genes found elsewhere in the plant genome was constructed. The result of the genome characterization and comparison showed that the genome characteristics, the gene composition, and the gene sequence of the chloroplast genes varied by location, and the gene regulatory network showed that the differences in growth location may have led to variations in the protein-coding chloroplast gene via various metabolic processes. These findings provide new insights into the relationship between chloroplasts and the sensitive metabolism of C. argyrophylla and provide additional reference materials for the conservation of this endangered plant.

In silico-guided metabolic engineering of Bacillus subtilis for efficient biosynthesis of purine nucleosides by blocking the key backflow nodes.

BACKGROUND: Purine nucleosides play essential roles in cellular physiological processes and have a wide range of applications in the fields of antitumor/antiviral drugs and food. However, microbial overproduction of purine nucleosides by de novo metabolic engineering remains a great challenge due to their strict and complex regulatory machinery involved in biosynthetic pathways. RESULTS: In this study, we designed an in silico-guided strategy for overproducing purine nucleosides based on a genome-scale metabolic network model in Bacillus subtilis. The metabolic flux was analyzed to predict two key backflow nodes, Drm (purine nucleotides toward PPP) and YwjH (PPP-EMP), to resolve the competitive relationship between biomass and purine nucleotide synthesis. In terms of the purine synthesis pathway, the first backflow node Drm was inactivated to block the degradation of purine nucleotides, which greatly increased the inosine production to 13.98-14.47 g/L without affecting cell growth. Furthermore, releasing feedback inhibition of the purine operon by promoter replacement enhanced the accumulation of purine nucleotides. In terms of the central carbon metabolic pathways, the deletion of the second backflow node YwjH and overexpression of Zwf were combined to increase inosine production to 22.01 ± 1.18 g/L by enhancing the metabolic flow of PPP. By switching on the flux node of the glucose-6-phosphate to PPP or EMP, the final inosine engineered strain produced up to 25.81 ± 1.23 g/L inosine by a pgi-based metabolic switch with a yield of 0.126 mol/mol glucose, a productivity of 0.358 g/L/h and a synthesis rate of 0.088 mmol/gDW/h, representing the highest yield in de novo engineered inosine bacteria. Under the guidance of this in silico-designed strategy, a general chassis bacterium was generated, for the first time, to efficiently synthesize inosine, adenosine, guanosine, IMP and GMP, which provides sufficient precursors for the synthesis of various purine intermediates. CONCLUSIONS: Our study reveals that in silico-guided metabolic engineering successfully optimized the purine synthesis pathway by exploring efficient targets, which could be applied as a superior strategy for efficient biosynthesis of biotechnological products.

A Universal Method for Developing Autoinduction Expression Systems Using AHL-Mediated Quorum-Sensing Circuits.

A major challenge in engineering microorganisms for the desirable product is maintaining the rational balance between cell growth and production. Quorum sensing (QS)-based dynamic regulations provide a pathway-independent genetic control technology to rebalance metabolic flux for biomass and product synthesis. However, the lack of a universal method for screening QS elements and the complex design of autoinduction circuits limit their applications in metabolic engineering. Here, we developed a universal method for simple and rapid screening and evaluating various QS systems from Gram-negative bacteria, and the largest library containing 195 combinations of receiving device/signal molecules was constructed and evaluated in Escherichia coli. A simple logical circuit with different inducer synthesis rates was established to dynamically regulate gene expression levels, leading to efficient protein expression and product synthesis. The system was further applied in Pseudomonas putida, which indicated it could be widely accommodated in other microorganisms. Therefore, the method could be used in diverse Gram-negative strains for the desired biosynthesis.

A mRNA Vaccine Encoding for a RBD 60-mer Nanoparticle Elicits Neutralizing Antibodies and Protective Immunity Against the SARS-CoV-2 Delta Variant in Transgenic K18-hACE2 Mice.

Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the Delta variant elicits robust neutralizing antibody response, and also provides protective immunity against the Delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern.

Adjunctive interleukin-2 for the treatment of drug-susceptible tuberculosis: a randomized control trial in China.

PURPOSE: Evaluation of the efficacy and safety of IL-2 in the treatment of drug-susceptible tuberculosis. METHODS: First, the cases of diagnosed drug-susceptible tuberculosis were randomized into two groups-the control group that received the background regimen of isoniazid, rifampin, pyrazinamide, and ethambutol, and the experimental group that received the background regimen plus IL-2. The efficacy and safety evaluations were performed throughout the therapy process as well as 12 months after the treatment completion. RESULTS: A total of 1151 patients underwent the randomization, among which 539 (96.2%) of the 560 in the experimental group achieved the sputum culture conversion to negative, compared to the 551 (93.2%) of the 591 in the control group, after 2 months of treatment, with significant difference observed between the groups (P = 0.025). Cavity closure after 2 months in the IL-2 (experimental) group was 60/211 (28.4%) compared to 46/248 (18.5%) in the control group, with a significant difference between the groups (P = 0.001). After treatment completion, the proportion of favorable outcomes was 559/560 (99.8%) in the experimental group and 587/591 (99.3%) in the control group, with no significant difference between the groups. Twelve months after treatment completion, relapse occurred in 15/560 (2.6%) in the IL-2 group and 19/591 (3.2%) in the control group, with no significant difference. CONCLUSION: IL-2 may enhance culture conversion and the cavity closure rate in the early treatment phase, although the enhancement may not be significant after treatment completion.

Simultaneous Multiplex Genome Engineering via Accelerated Natural Transformation in Bacillus subtilis.

Multiplex engineering at the scale of whole genomes has become increasingly important for synthetic biology and biotechnology applications. Although several methods have been reported for engineering microbe genomes, their use is limited by their complex procedures using multi-cycle transformations. Natural transformation, involving in species evolution by horizontal gene transfer in many organisms, indicates its potential as a genetic tool. Here, we aimed to develop simultaneous multiplex genome engineering (SMGE) for the simple, rapid, and efficient design of bacterial genomes via one-step of natural transformation in Bacillus subtilis. The transformed DNA, competency factors, and recombinases were adapted to improved co-editing frequencies above 27-fold. Single to octuplet variants with genetic diversity were simultaneously generated using all-in-one vectors harboring multi-gene cassettes. To demonstrate its potential application, the tyrosine biosynthesis pathway was further optimized for producing commercially important resveratrol by high-throughput screening of variant pool in B. subtilis. SMGE represents an accelerated evolution platform that generates diverse multiplex mutations for large-scale genetic engineering and synthetic biology in B. subtilis.

Comparative Evaluation of Bandage Contact Lenses and Eye Patching after Bilateral Cataract Surgery.

PURPOSE: To comparatively evaluate the safety and satisfaction of bandage contact lens (BCL) and eye patching in patients after cataract surgery. METHODS: Sixteen (32 eyes) patients who planned to undergo bilateral cataract surgery were recruited. The two eyes of each patient were randomly divided into 2 groups. Group A and Group B were instructed to wear BCLs immediately at the end of the surgery until one week and eye patch immediately after surgery until one day, respectively. Visual analog scales of ten specific symptoms, Visual Function Index (VF-14) questionnaire, and best-corrected visual acuity (BCVA) were conducted on the first day before the surgery and Day 1 and Day 7 after surgery. Oculus keratography was conducted on the first day before surgery and on Day 7. Patient satisfaction was determined on Day 1. Moreover, bacterial species in the conjunctival sac, meibomian gland secretions, and BCLs were subsequently identified using 16S rRNA gene sequencing. RESULTS: The patient satisfaction scores of Group A were higher than Group B. Group A were more motivated to choose the same treatment and were more likely to recommend BCLs to others. No statistically significant differences were found in bacterial culture positivity between the groups. The differences in ocular signs and symptoms between the two groups were not statistically significant. There were no significant differences in the BCVA and VF-14 between the groups at any time point. CONCLUSIONS: BCLs could be safely and effectively used in patients after cataract surgery.

Use of comedications and potential drug-drug interactions in people living with HIV in China.

BACKGROUND: Because people living with HIV (PLWH) are ageing, they will inevitably develop non-communicable diseases (NCDs) and the number of non-HIV medications will increase. Drug-drug interactions(DDIs) will become an ever-increasing issue. However, little is known about this important issue in Chinese PLWH. This study aimed to investigate the prevalence and risk factors of DDIs among PLWH in China. METHODS: Chinese PLWH aged ≥18 years were enrolled prospectively from October 2018 to April 2019 and after informed consent was obtained, they were ask to fill out a questionnaire about comorbidity and co-medications. Potential DDIs were identified using the University of Liverpool HIV Drug Interaction Checker. RESULTS: A total of 1804 questionnaires were included. Antiretroviral drugs (ARVs) that most frequently were prescribed were lamivudine (96.18%), efavirenz(64.64%) and tenofovir(60.62%). 16.96% of the participations reported current co-infection with HIV and14.69% reported NCDs. 263(14.57%) participations reported they had used co-medications in the past six months while 186(10.31%) reported they were taking co-medications. Age≥50 years (p < 0.001), living in developed areas(p < 0.001) and lower CD4 cell count(p = 0.045) were independently associated with the use of co-medications. Potential DDIs were identified in 54 (19.15%) persons using co-medications. Age≥50 [OR = 2.272(1.241-4.158)], PLWH with NCDs[OR = 2.889(1.509-5.532)] and usage of protease inhibitors[OR = 2.538(1.250-5.156)] were independently associated with the potential DDIs. CONCLUSION: The prevalence of the use of co-medications and potential DDIs among Chinese PLWH are low. Older age, NCDs and use of PIs were risk factors for the potential of developing DDIs. With the aging of PLWH, co-medications and DDIs in China warrants more attention.

Lysobacter oculi sp. nov., isolated from human Meibomian gland secretions.

A Gram-stain negative, rod-shaped bacterial, catalase and oxidase positive strain (83-4T) that formed yellow colonies was isolated from human Meibomian gland secretions. Strain 83-4T belongs to the genus Lysobacter according to phylogenetic analysis based on 16S rRNA gene sequences. The DNA G+C content was 67.1 mol%. The circular genome was 2.6 Mb, which contained 2431 protein-coding sequences, 75 pseudogenes, 46 tRNAs, 3 rRNAs and 4 ncRNAs. A bacteriocin cluster and aryl polyene cluster were also found in the genome. The average nucleotide identity value was 79.6% between isolate 83-4T and the closely related type strain Lysobacter tolerans UM1T. The estimated DNA-DNA hybridization value between strain 83-4T and L. tolerans UM1T was 41.6%. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids. Iso-C15:0, iso-C11:0 3-OH, iso-C11:0 and summed feature 9 (iso-C17:1ω9c) were the major fatty acids. Ubiquinone (Q-8) was the only respiratory quinone. Therefore, based on the data of phylogenetic analysis, chemotaxonomical and biochemical analyses, it is concluded that strain 83-4T represents a novel species of the genus Lysobacter with the name of Lysobacter oculi sp. nov. The type strain is 83-4T (= CGMCC 1.13464T = NRBC 113451T).

A seamless and iterative DNA assembly method named PS-Brick and its assisted metabolic engineering for threonine and 1-propanol production.

BACKGROUND: DNA assembly is an essential technique enabling metabolic engineering and synthetic biology. Combining novel DNA assembly technologies with rational metabolic engineering can facilitate the construction of microbial cell factories. Amino acids and derived biochemicals are important products in industrial biotechnology with wide application and huge markets. DNA assembly scenarios encountered in metabolic engineering for the construction of amino acid and related compound producers, such as design-build-test-learn cycles, construction of precise genetic circuits and repetitive DNA molecules, usually require for iterative, scarless and repetitive sequence assembly methods, respectively. RESULTS: Restriction endonuclease (RE)-assisted strategies constitute one of the major categories of DNA assembly. Here, we developed a Type IIP and IIS RE-assisted method named PS-Brick that comprehensively takes advantage of the properties of PCR fragments and REs for iterative, seamless and repetitive sequence assembly. One round of PS-Brick reaction using purified plasmids and PCR fragments was accomplished within several hours, and transformation of the resultant reaction product from this PS-Brick assembly reaction exhibited high efficiency (104-105 CFUs/µg DNA) and high accuracy (~ 90%). An application of metabolic engineering to threonine production, including the release of feedback regulation, elimination of metabolic bottlenecks, intensification of threonine export and inactivation of threonine catabolism, was stepwise resolved in E. coli by rounds of "design-build-test-learn" cycles through the iterative PS-Brick paradigm, and 45.71 g/L threonine was obtained through fed-batch fermentation. In addition to the value of the iterative character of PS-Brick for sequential strain engineering, seamless cloning enabled precise in-frame fusion for codon saturation mutagenesis and bicistronic design, and the repetitive sequence cloning ability of PS-Brick enabled construction of tandem CRISPR sgRNA arrays for genome editing. Moreover, the heterologous pathway deriving 1-propanol pathway from threonine, composed of Lactococcus lactis kivD and Saccharomyces cerevisiae ADH2, was assembled by one cycle of PS-Brick, resulting in 1.35 g/L 1-propanol in fed-batch fermentation. CONCLUSIONS: To the best of our knowledge, the PS-Brick framework is the first RE-assisted DNA assembly method using the strengths of both Type IIP and IIS REs. In this study, PS-Brick was demonstrated to be an efficient DNA assembly method for pathway construction and genome editing and was successfully applied in design-build-test-learn (DBTL) cycles of metabolic engineering for the production of threonine and threonine-derived 1-propanol. The PS-Brick presents a valuable addition to the current toolbox of synthetic biology and metabolic engineering.