Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination.

Co-option of RAG1 and RAG2 for antigen receptor gene assembly by V(D)J recombination was a crucial event in the evolution of jawed vertebrate adaptive immunity. RAG1/2 are proposed to have arisen from a transposable element, but definitive evidence for this is lacking. Here, we report the discovery of ProtoRAG, a DNA transposon family from lancelets, the most basal extant chordates. A typical ProtoRAG is flanked by 5-bp target site duplications and a pair of terminal inverted repeats (TIRs) resembling V(D)J recombination signal sequences. Between the TIRs reside tail-to-tail-oriented, intron-containing RAG1-like and RAG2-like genes. We demonstrate that ProtoRAG was recently active in the lancelet germline and that the lancelet RAG1/2-like proteins can mediate TIR-dependent transposon excision, host DNA recombination, transposition, and low-efficiency TIR rejoining using reaction mechanisms similar to those used by vertebrate RAGs. We propose that ProtoRAG represents a molecular "living fossil" of the long-sought RAG transposon.

Structures and mechanisms of the Arabidopsis auxin transporter PIN3.

The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development1,2. Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thaliana PIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static.

Honeycomb-Structured MoSe2 /rGO Composites as High-Performance Anode Materials for Sodium-Ion Batteries.

Sodium-ion batteries are a promising substitute for lithium batteries due to the abundant resources and low cost of sodium. Herein, honeycomb-shaped MoSe2 /reduced graphene oxide (rGO) composite materials are synthesized from graphene oxide (GO) and MoSe2 through a one-step solvothermal process. Experiments show that the 3D honeycomb structure provides excellent electrolyte penetration while alleviating the volume change during electrochemical cycling. An anode prepared with MoSe2 /rGO composites exhibits significantly improved sodium-ion storage properties, where a large reversible capacity of 215 mAh g-1 is obtained after 2700 cycles at the current density of 30.0 A g-1 or after 5900 cycles at 8.0 A g-1 . When such an anode is paired with Na3 V2 (PO4 )3 to form a full cell, a reversible specific capacity of 107.5 mAh g-1 can be retained after 1000 cycles at the current of 1.0 A g-1 . Transmission electron microscopy, X-ray photoelectron spectroscopy and in situ X-ray diffraction (XRD) characterization reveal the reversible storage reaction of Na ions in the MoSe2 /rGO composites. The significantly enhanced sodium storage capacity is attributed to the unique honeycomb microstructure and the use of ether-based electrolytes. This study illustrates that combining rGO with ether-based electrolytes has tremendous potential in constructing high-performance sodium-ion batteries.

Addition of rapamycin or co-culture with cumulus cells from younger reproductive age women does not improve rescue in vitro oocyte maturation or euploidy rates in older reproductive age women.

Both spontaneously conceived pregnancies and those achieved using assisted reproduction decline with advancing maternal age. In this study, we tested if rapamycin and/or cumulus cells (CCs) from young donors could improve oocyte maturation and euploidy rates of germinal vesicle (GV) stage oocytes obtained from older women of reproductive age. A total of 498 GVs from 201 women >38 years (40.6 ± 1.8, mean ± SD) were included. GVs were randomly assigned into five groups for rescue IVM: control (with no CCs and no rapamycin); with autologous CCs; with autologous CCs and rapamycin; with CCs from young women (<35 years); and with CCs from young women and rapamycin. After 24 h of culture, the first polar body (PB) was biopsied in metaphase II oocytes, and the cytogenetic constitution was assessed using next-generation sequencing for both oocytes and PBs. Comparable maturation rates were found (56.2%, 60.0%, 46.5%, 51.7%, and 48.5% for groups 1-5, respectively; P = 0.30). Similarly, comparable euploidy rates were observed in the five groups (41.5%, 37.8%, 47.2%, 43.6%, and 47.8% for Groups 1-5, respectively; P = 0.87). Our findings indicate that rescue IVM is effective for obtaining mature euploid oocytes in older women of reproductive age, and that incubation with rapamycin or CCs obtained from young donors does not improve the maturation or euploidy rate.

ExpressVis: a biologist-oriented interactive web server for exploring multi-omics data.

In the era of life-omics, huge amounts of multi-omics data have been generated and widely used in biomedical research. It is challenging for biologists with limited programming skills to obtain biological insights from multi-omics data. Thus, a biologist-oriented platform containing visualization functions is needed to make complex omics data digestible. Here, we propose an easy-to-use, interactive web server named ExpressVis. In ExpressVis, users can prepare datasets; perform differential expression analysis, clustering analysis, and survival analysis; and integrate expression data with protein-protein interaction networks and pathway maps. These analyses are organized into six modules. Users can use each module independently or use several modules interactively. ExpressVis displays analysis results in interactive figures and tables, and provides comprehensive interactive operations in each figure and table, between figures or tables in each module, and among different modules. It is freely accessible at https://omicsmining.ncpsb.org.cn/ExpressVis and does not require login. To test the performance of ExpressVis for multi-omics studies of clinical cohorts, we re-analyzed a published hepatocellular carcinoma dataset and reproduced their main findings, suggesting that ExpressVis is convenient enough to analyze multi-omics data. Based on its complete analysis processes and unique interactive operations, ExpressVis provides an easy-to-use solution for exploring multi-omics data.

Environmental TEM Study of the Dispersion of Au/α-MoC: From Nanoparticles to Two-Dimensional Clusters.

The synthesis of highly dispersed Au nanoclusters that are stable under elevated temperatures in heterogeneous catalysis is challenging. Here, we directly observe a strong metal-support interaction (SMSI)-induced dispersion of Au nanoparticles (NPs) on α-MoC using an environmentally atomically resolved secondary imaging technique. Under a realistic environment, Au NPs flatten and spread out on the α-MoC to form two-dimensional atomic layered clusters. The formed highly dispersed Au/α-MoC catalyst shows excellent stability at 600 °C for 160 h in the reverse water-gas shift reaction. The X-ray photoelectron spectrum and extended X-ray absorption fine structure results show that Au NPs gradually become low-coordination-number cluster species and lose electrons to become Auδ+; these form chemical bonds with the α-MoC support and are responsible for the dispersion behavior. This work provides an insightful understanding of dispersion behavior and promotes the rational design and synthesis of reverse sintering catalysts.

Rationally Designed Cyclooctatetrathiophene-Based Porous Aromatic Frameworks (COTh-PAFs) for Efficient Photocatalytic Hydrogen Peroxide Production.

Constructing stable and efficient photocatalysts for H2O2 production is of great importance and is challenging. In this study, the synthesis of three photoactive cyclooctatetrathiophene (COTh)-based porous aromatic frameworks (COTh-PAFs) in an alternating donor-acceptor (D-A) fashion is presented. In combination with a triazine-derived electron acceptor, PAF-363 exhibits high efficiency for the photosynthesis of H2O2 with production rates of 11733 µmol g-1 h-1(with sacrificial agent) and 3930 µmol g-1 h-1 (without sacrificial agent) from water and oxygen under visible light irradiation. Experimental results and theoretical calculations reveal that the charge transfer positions and the O2 adsorption sites in PAF-363 are both concentrated on COTh fragments, which facilitate the H2O2 production through the oxygen reduction reaction (ORR) pathway. This work highlights that the rational design of COTh-PAFs with consideration of D-A direction, charge transfer positions, and O2 adsorption sites provides a feasible access to efficient H2O2 production photocatalysts.

Covalent Organic Framework Nanohydrogels.

Nanohydrogelation of covalent organic frameworks (COFs) will undoubtedly open up new applications for them in water, such as aqueous catalysis and biomedicine. It is currently a great challenge to achieve water dispersion of COFs through either bottom-up construction strategies or top-down exfoliating technologies. Herein, poly(N-isopropylacrylamide) (PNIPAM)-postmodified COF nanohydrogels (COF-NHGs) are successfully designed and synthesized via in situ atom-transfer radical polymerization (ATRP) on a scaffold of COFs. During the polymer growth process, the bulk COFs are exfoliated into nanosheets with a lateral size of ∼500 nm and a thickness of ∼6.5 nm. Moreover, their size can be precisely controlled by the degree of polymerization of PNIPAMs. In aqueous solution, the obtained COF-NHGs are assembled into nanohydrogels retaining intra-plane crystallinity and exhibit a temperature-sensitive sol-gel phase transition. With excellent solubility in organic solvents, the COF-NHGs' intrinsic physical properties in the solution state can be characterized through their solution nuclear magnetic resonance and ultraviolet absorption spectra. These results put forward new opportunities for regulating the solution processability of COFs and building an intelligent, stimuli-response platform of COF-polymer composite nanohydrogels for device applications.

MOF-Derived Ru1Zr1/Co Dual-Atomic-Site Catalyst with Promoted Performance for Fischer-Tropsch Synthesis.

Cobalt-based catalysts have been widely used for Fischer-Tropsch synthesis (FTS) in industry; however, achieving rational catalyst design at the atomic level and thereby a higher activity and more long-chain-hydrocarbon products simultaneously remain an attractive and difficult challenge. The dual-atomic-site catalysts with unique electronic and geometric interface interactions offer a great opportunity for exploiting advanced FTS catalysts with improved performance. Herein, we designed a Ru1Zr1/Co catalyst with Ru and Zr dual atomic sites on the Co nanoparticle (NP) surface through a metal-organic-framework-mediated synthesis strategy which presents greatly enhanced FTS activity (high turnover frequency of 3.8 × 10-2 s-1 at 200 °C) and C5+ selectivity (80.7%). Control experiments presented a synergic effect between Ru and Zr single-atom site on Co NPs. Further density functional theory calculations of the chain growth process from C1 to C5 revealed that the designed Ru/Zr dual sites remarkably lower the rate-limiting barriers due to the significantly weakened C-O bond and promote the chain growth processes, resulting in the greatly boosted FTS performance. Therefore, our work demonstrates the effectiveness of dual-atomic-site design in promoting the FTS performance and provides new opportunities for developing efficient industrial catalysts.

A Coronary CT Angiography Radiomics Model to Identify Vulnerable Plaque and Predict Cardiovascular Events.

Background A noninvasive coronary CT angiography (CCTA)-based radiomics technique may facilitate the identification of vulnerable plaques and patients at risk for future adverse events. Purpose To assess whether a CCTA-based radiomic signature (RS) of vulnerable plaques defined with intravascular US was associated with increased risk for future major adverse cardiac events (MACE). Materials and Methods In a retrospective study, an RS of vulnerable plaques was developed and validated using intravascular US as the reference standard. The RS development data set included patients first undergoing CCTA and then intravascular US within 3 months between June 2013 and December 2020 at one tertiary hospital. The development set was randomly assigned to training and validation sets at a 7:3 ratio. Diagnostic performance was assessed internally and externally from three tertiary hospitals using the area under the curve (AUC). The prognostic value of the RS for predicting MACE was evaluated in a prospective cohort with suspected coronary artery disease between April 2018 and March 2019. Multivariable Cox regression analysis was used to evaluate the RS and conventional anatomic plaque features (eg, segment involvement score) for predicting MACE. Results The RS development data set included 419 lesions from 225 patients (mean age, 64 years ± 10 [SD]; 68 men), while the prognostic cohort included 1020 lesions from 708 patients (mean age, 62 years ± 11; 498 men). Sixteen radiomic features, including two shape features and 14 textural features, were selected to build the RS. The RS yielded a moderate to good AUC in the training, validation, internal, and external test sets (AUC = 0.81, 0.75, 0.80, and 0.77, respectively). A high RS (≥1.07) was independently associated with MACE over a median 3-year follow-up (hazard ratio, 2.01; P = .005). Conclusion A coronary CT angiography-derived radiomic signature of coronary plaque enabled the detection of vulnerable plaques that were associated with increased risk for future adverse cardiac outcomes. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by De Cecco and van Assen in this issue.

Evolocumab attenuate pericoronary adipose tissue density via reduction of lipoprotein(a) in type 2 diabetes mellitus: a serial follow-up CCTA study.

BACKGROUND: Pericoronary adipose tissue (PCAT) density is a biomarker of vessel inflammation, which is supposed to be increased in patients with type 2 diabetes mellitus (T2DM). However, whether the coronary inflammation revealed by this novel index could be alleviated after evolocumab treatment in T2DM remains unknown. METHODS: From January 2020 to December 2022, consecutive T2DM patients with low-density lipoprotein cholesterol ≥ 70 mg/dL on maximally tolerated statin and taking evolocumab were prospectively included. In addition, patients with T2DM who were taking statin alone were recruited as control group. The eligible patients underwent baseline and follow-up coronary CT angiography with an interval of 48-week. To render patients with evolocumab as comparable to those controls, a propensity-score matching design was used to select the matched pairs with a 1:1 ratio. Obstructive lesion was defined as the extent of coronary artery stenosis ≥ 50%; the numbers inside the brackets were interquartile ranges. RESULTS: A total of 170 T2DM patients with stable chest pain were included [(mean age 64 ± 10.6 [range 40-85] years; 131 men). Among those patients, 85 were in evolocumab group and 85 were in control group. During follow-up, low-density lipoprotein cholesterol (LDL-C) level (2.02 [1.26, 2.78] vs. 3.34 [2.53, 4.14], p < 0.001), and lipoprotein(a) (12.1 [5.6, 21.8] vs. 18.9 [13.2, 27.2], p = 0.002) were reduced after evolocumab treatment. The prevalence of obstructive lesions and high-risk plaque features were significantly decreased (p < 0.05 for all). Furthermore, the calcified plaque volume were significantly increased (188.3 [115.7, 361.0] vs. 129.3 [59.5, 238.3], p = 0.015), while the noncalcified plaque volume and necrotic volume were diminished (107.5 [40.6, 180.6] vs. 125.0 [65.3, 269.7], p = 0.038; 0 [0, 4.7] vs. 0 [0, 13.4], p < 0.001, respectively). In addition, PCAT density of right coronary artery was significantly attenuated in evolocumab group (- 85.0 [- 89.0, - 82.0] vs. - 79.0 [- 83.5, - 74.0], p < 0.001). The change in the calcified plaque volume inversely correlated with achieved LDL-C level (r = - 0.31, p < 0.001) and lipoprotein(a) level (r = - 0.33, p < 0.001). Both the changes of noncalcified plaque volume and necrotic volume were positively correlated with achieved LDL-C level and Lp(a) (p < 0.001 for all). However, the change of PCATRCA density only positively correlated with achieved lipoprotein(a) level (r = 0.51, p < 0.001). Causal mediation analysis revealed Lp(a) level mediated 69.8% (p < 0.001) for the relationship between evolocumab and changes of PCATRCA. CONCLUSIONS: In patients with T2DM, evolocumab is an effective therapy to decrease noncalcified plaque volume necrotic volume, and increase calcified plaque volume. Furthermore, evolocumab could attenuate PCAT density, at least in part, via the reduction of lipoprotein(a).

The effect of luteinizing hormone changes in GnRH antagonist protocol on the outcome of controlled ovarian hyperstimulation and embryo transfer.

BACKGROUD: To investigate the effect of Luteinizing hormone (LH) level changes on the outcomes of controlled ovarian hyperstimulation (COH) and embryo transfer (ET) in gonadotropin-releasing hormone antagonist (GnRH-ant) protocol. METHODS: A total of 721 patients undergoing GnRH-ant protocol COH for the first IVF/ICSI cycles were retrospectively analyzed. COH process were divided into 2 stages, before (stage 1) and after (stage 2) the GnRH-ant initiation, and each with 5 groups basing on LH levels: LH decreased more than 50% (groups A1, A2), decreased 25-50% (groups B1, B2), change less than 25% (groups C1, C2), increased 25-50% (groups D1, D2), and increased more than 50% (groups E1, E2). RESULTS: There were no significant differences among groups of stage1 regarding COH and ET outcomes. For stage 2, the more obvious the decrease of LH level, the more the number of oocytes retrieved, mature oocytes, fertilized oocytes, embryos cleavaged and the numbers of embryo available (P < 0.05), but without significant differences regarding ET outcomes. We also found the freeze-all rate in Group A2 was higher (P < 0.001). CONCLUSION: LH level changes before GnRH-ant addition were not related to COH and ET outcomes. LH level changes after the addition of GnRH-ant were related to the outcome of COH, and no significant differences were found relating to ET outcomes.

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.

Clinical nutrition service capacity of 445 secondary or above hospitals in Sichuan, China: the 2021 annual survey.

BACKGROUND AND OBJECTIVES: To investigate the capacity of clinical nutrition services in secondary and tertiary hospitals in the Sichuan Province, China. METHODS AND STUDY DESIGN: Convenience sampling was used. E-questionnaires were distributed to all eligible medical institutions in Sichuan through the official network of provincial and municipal clinical nutrition quality control centers. The data obtained were sorted in Microsoft Excel and analyzed by SPSS. RESULTS: A total of 519 questionnaires were returned, of which 455 were valid. Only 228 hospitals were accessible to clinical nutrition services, of which 127 hospitals had independently set up clinical nutrition departments (CNDs). The ratio of clinical nutritionists to beds was 1:214. During the last decade, the rate of constructing new CNDs was maintained at approximately 5 units/year. A total of 72.4% of hospitals managed their clinical nutrition units as part of their medical technology departments. The specialist number ratio of senior, associate, intermediate and junior is approximately 1:4:8:10. There were 5 common charges for clinical nutrition. CONCLUSIONS: The sample representation was limited, and the capacity of clinical nutrition services may have been overestimated. Secondary and tertiary hospitals in Sichuan are currently in the second high tide of department establishment, with a positive trend of departmental affiliation standardization and a basic formation of a talent echelon.

Genome-Wide Analysis of Long Noncoding RNAs in Porcine Intestine during Weaning Stress.

Long noncoding RNAs (lncRNAs) play crucial roles in various biological processes, and they are considered to be closely associated with the pathogenesis of intestinal diseases. However, the role and expression of lncRNAs in intestinal damage during weaning stress remain unknown. Herein, we investigated the expression profiles of jejunal tissue from weaning piglets at 4 and 7 d after weaning (groups W4 and W7, respectively) and from suckling piglets on the same days (groups S4 and S7, respectively). Genome-wide analysis of lncRNAs was also performed using RNA sequencing technology. A total of 1809 annotated lncRNAs and 1612 novel lncRNAs were obtained from the jejunum of piglets. In W4 vs. S4, a total of 331 lncRNAs showed significant differential expression, and a total of 163 significantly differentially expressed lncRNAs (DElncRNAs) was identified in W7 vs. S7. Biological analysis indicated that DElncRNAs were involved in intestinal diseases, inflammation, and immune functions, and were mainly enriched in the Jak-STAT signaling pathway, inflammatory bowel disease, T cell receptor signaling pathway, B cell receptor signaling pathway and intestinal immune network for IgA production. Moreover, we found that lnc_000884 and target gene KLF5 were significantly upregulated in the intestine of weaning piglets. The overexpression of lnc_000884 also significantly promoted the proliferation and depressed apoptosis of IPEC-J2 cells. This result suggested that lnc_000884 may contribute to repairing intestinal damage. Our study identified the characterization and expression profile of lncRNAs in the small intestine of weaning piglets and provided new insights into the molecular regulation of intestinal damage during weaning stress.

Induced Pluripotent Stem Cells for Tissue-Engineered Skeletal Muscles.

Skeletal muscle, which comprises a significant portion of the body, is responsible for vital functions such as movement, metabolism, and overall health. However, severe injuries often result in volumetric muscle loss (VML) and compromise the regenerative capacity of the muscle. Tissue-engineered muscles offer a potential solution to address lost or damaged muscle tissue, thereby restoring muscle function and improving patients' quality of life. Induced pluripotent stem cells (iPSCs) have emerged as a valuable cell source for muscle tissue engineering due to their pluripotency and self-renewal capacity, enabling the construction of tissue-engineered artificial skeletal muscles with applications in transplantation, disease modelling, and bio-hybrid robots. Next-generation iPSC-based models have the potential to revolutionize drug discovery by offering personalized muscle cells for testing, reducing reliance on animal models. This review provides a comprehensive overview of iPSCs in tissue-engineered artificial skeletal muscles, highlighting the advancements, applications, advantages, and challenges for clinical translation. We also discussed overcoming limitations and considerations in differentiation protocols, characterization methods, large-scale production, and translational regulations. By tackling these challenges, iPSCs can unlock transformative advancements in muscle tissue engineering and therapeutic interventions for the future.

Predicting embryonic aneuploidy rate in IVF patients using whole-exome sequencing.

Infertility is a major reproductive health issue that affects about 12% of women of reproductive age in the United States. Aneuploidy in eggs accounts for a significant proportion of early miscarriage and in vitro fertilization failure. Recent studies have shown that genetic variants in several genes affect chromosome segregation fidelity and predispose women to a higher incidence of egg aneuploidy. However, the exact genetic causes of aneuploid egg production remain unclear, making it difficult to diagnose infertility based on individual genetic variants in mother's genome. In this study, we evaluated machine learning-based classifiers for predicting the embryonic aneuploidy risk in female IVF patients using whole-exome sequencing data. Using two exome datasets, we obtained an area under the receiver operating curve of 0.77 and 0.68, respectively. High precision could be traded off for high specificity in classifying patients by selecting different prediction score cutoffs. For example, a strict prediction score cutoff of 0.7 identified 29% of patients as high-risk with 94% precision. In addition, we identified MCM5, FGGY, and DDX60L as potential aneuploidy risk genes that contribute the most to the predictive power of the model. These candidate genes and their molecular interaction partners are enriched for meiotic-related gene ontology categories and pathways, such as microtubule organizing center and DNA recombination. In summary, we demonstrate that sequencing data can be mined to predict patients' aneuploidy risk thus improving clinical diagnosis. The candidate genes and pathways we identified are promising targets for future aneuploidy studies.

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.

Discovery and preclinical evaluations of JBD0131, a novel nitrodihydro-imidazooxazole anti-tuberculosis agent.

Multidrug-resistant pulmonary tuberculosis (MDR-TB) is a major health problem worldwide. The treatment for MDR-TB requires medications for a long duration (up to 20-24 months) with second-line drugs resulting in unfavorable outcomes. Nitroimidazoles are promising antimycobacterial agents known to inhibit both aerobic and anaerobic mycobacterial activity. Delamanid and pretomanid are two nitroimidazoles approved by the regulatory agencies for MDR-TB treatment. However, both agents possess unsatisfactory absorption and QTc prolongation. In our search for a safer nitroimidazole, we discovered JBD0131 (2). It exhibited excellent anti-mycobacterial activity against M. tuberculosis H37Rv in vitro and in vivo, improved PK and absorption, reduced QT prolongation potential of delamanid. JBD0131 is currently in clinical development in China for pulmonary tuberculosis (CTR20202308).