Discovery and Heterologous Production of Tetrapetalones Provide Insights into the Formation of the Tetracyclic System.

Tetrapetalones make up a unique class of pentaketide ansamycins that feature a tetracyclic skeleton and exhibit potent inhibitory activities against soybean lipoxygenase. However, a detailed biosynthetic route to tetrapetalones has not been published. Herein we report the activation of the tetrapetalones' biosynthetic gene cluster (tpt) in Streptomyces sp. S10 by promoter engineering along with constitutive expression of pathway-specific regulator genes, leading to the discovery of seven new derivatives, tetrapetalones E-K (2-8), and the known tetrapetalone A (1). In vivo gene deletion experiments and heterologous expression of the minimized tpt cluster in Streptomyces albus J1074 suggest that the tetracyclic system of tetrapetalones is probably formed spontaneously, and the regioselective glycosylation of tetrapetalones at the C-9 hydroxy group with d-rhamnose or d-rhodinose was catalyzed by the glycosyltransferase Tpt14.

Efficient and Stable Tin-Lead Mixed Perovskite Solar Cells Using Post-Treatment Additive with Synergistic Effects.

Inhibiting the oxidation of Sn2+ during the crystallization process of Sn-Pb mixed perovskite film is found to be as important as the oxidation resistance of precursor solution to achieve high efficiency, but less investigated. Considering the excellent reduction feature of hydroquinone and the hydrophobicity of tert-butyl group, an antioxidant 2,5-di-tert-butylhydroquinone (DBHQ) was introduced into Sn-Pb mixed perovskite films using an anti-solvent approach to solve this problem. Interestingly, we find that DBHQ can act as function alterable additive during its utilization. On the one hand, DBHQ can restrict the oxidation of Sn2+ during the crystallization process, facilitating the fabrication of high-quality perovskite film; on the other hand, the generated oxidation product 2,5-di-tert-butyl-1,4-benzoquinone (DBBQ) can functionalize as defect passivator to inhibit the charge recombination. As a result, this synergetic effect renders the Sn-Pb mixed PSC a power conversion efficiency (PCE) up to 23.0 %, which is significantly higher than the reference device (19.6 %). Furthermore, the unencapsulated DBQH-modified PSCs exhibited excellent long-term stability and thermal stability, with the devices maintaining 84.2 % and 78.9 % of the initial PCEs after aging at 25 °C and 60 °C for 800 h and 120 h under N2 atmosphere, respectively. Therefore, the functional alterable strategy provides a novel cornerstone for high-performance Sn-Pb mixed PSCs.

Follistatin (FST) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.

Follistatin (FST), a member of the transforming growth factor-ß (TGF-ß) superfamily, has been identified as an inhibitor of follicle-stimulating hormone. Previous studies showed that it plays an important role in animal reproduction. Therefore, this study aims to investigate its effect on the maturation of buffalo oocytes in vitro, and the underlying mechanism of FST affecting oocyte maturation was also explored in buffalo cumulus cells. Results showed that FST was enriched in the ovary and expressed at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. The FST expression level was up-regulated in MII buffalo oocytes compared with the GV stage (p < .05). To study the effects of FST on buffalo oocytes' maturation and early embryonic development, we added the pcD3.1 skeleton vector and PCD3.1-EGFP-FST vector into the maturation fluid of buffalo oocytes, respectively. It was demonstrated that FST promoted the in vitro maturation rate of buffalo oocytes and the blastocyst rate of embryos cultured in vitro (p < .05). By interfering with FST expression, we discovered that FST in cumulus cells plays a crucial role in oocyte maturation. Interference with the FST expression during the buffalo oocyte maturation did not affect the first polar body rate of buffalo oocyte (p > .05). In contrast, the location of mitochondria in oocytes was abnormal, and the cumulus expansion area was reduced (p < .05). After parthenogenetic activation, the cleavage and blastocyst rates of the FST-interfered group were reduced (p < .05). Furthermore, RT-qPCR was performed to investigate further the underlying mechanism by which FST enhances oocyte maturation. We found that overexpression of FST could up-regulate the expression level of apoptosis suppressor gene Bcl-2 and TGF-ß/SMAD pathway-related genes TGF-ß, SMAD2, and SMAD3 (p < .05). In contrast, the expression levels of SMAD4 and pro-apoptotic gene BAX were significantly decreased (p < .05). The FST gene could affect buffalo oocyte maturation by regulating the oocyte mitochondria integrity, the cumulus expansion, cumulus cell apoptosis, and the expression levels of TGF-ß/SMAD pathway-related genes.

[A new xanthone from hulls of Garcinia mangostana and its cytotoxic activity].

Eight compounds were isolated from ethyl acetate fraction of 80% ethanol extract of the hulls of Garcinia mangostana by silica gel, Sephadex LH-20 column chromatography, as well as prep-HPLC methods. By HR-ESI-MS, MS, 1D and 2D NMR spectral analyses, the structures of the eight compounds were identified as 16-en mangostenone E(1), α-mangostin(2), 1,7-dihydroxy-2-(3-methy-lbut-2-enyl)-3-methoxyxanthone(3), cratoxyxanthone(4), 2,6-dimethoxy-para-benzoquinone(5), methyl orselinate(6), ficusol(7), and 4-(4-carboxy-2-methoxyphenoxy)-3,5-dimethoxybenzoic acid(8). Compound 1 was a new xanthone, and compound 4 was a xanthone dimer, compound 5 was a naphthoquinone. All compounds were isolated from this plant for the first time except compounds 2 and 3. Cytotoxic bioassay suggested that compounds 1, 2 and 4 possessed moderate cytotoxicity, suppressing HeLa cell line with IC_(50) va-lues of 24.3, 35.5 and 17.1 µmol·L~(-1), respectively. Compound 4 also could suppress K562 cells with an IC_(50) value of 39.8 µmol·L~(-1).

[Chemical constituents from Salacia polysperma].

Nine compounds were isolated from the 90% ethanol extract of Salacia polysperma by silica gel, Sephadex LH-20 column chromatography, together with preparative HPLC methods. Based on HR-ESI-MS, MS, 1D and 2D NMR spectral analyses, the structures of the nine compounds were identified as 28-hydroxy wilforlide B(1), wilforlide A(2), 1ß,3ß-dihydroxyurs-9(11),12-diene(3),(-)-epicatechin(4),(+)-catechin(5),(-)-4'-O-methyl-ent-galloepicatechin(6), 3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)propan-1-one(7),(-)-(7S,8R)-4-hydroxy-3,3',5'-trimethoxy-8',9'-dinor-8,4'-oxyneoligna-7,9-diol-7'-aldehyde(8), and vanillic acid(9). Compound 1 is a new oleanane-type triterpene lactone. Compounds 1, 3, 4, 7-9 were isolated from the Salacia genus for the first time. All compounds were assayed for their α-glucosidase inhibitory activity. The results suggested that compound 8 exhibited moderate α-glucosidase inhibitory activity, with an IC_(50) value of 37.2 µmol·L~(-1), and the other compounds showed no α-glucosidase inhibitory activity.

Identification and Characterization of the 28-N-Methyltransferase Involved in HSAF Analogue Biosynthesis.

Polycyclic tetramate macrolactams (PoTeMs) are a family of structurally intriguing bioactive natural products. Although the presence of the N-28 methyl group is known to affect bioactivities of some PoTeMs, the mechanism for this methylation remains unclear. We report here the identification and characterization of the 28-N-methyltransferase for HSAF analogues, which is encoded by a gene located outside the HSAF (heat-stable antifungal factor) cluster in Lysobacter enzymogenes C3. Our data suggested that 28-N-methyltransferase utilizes S-adenosylmethionine (SAM) to methylate HSAF analogues, and acts after the dicyclic and tricyclic ring formation and prior to C-3 hydroxylation. Kinetic analysis showed that the optimal substrate for the enzyme is 3-dehydroxy HSAF (3-deOH HSAF). Moreover, it could also accept PoTeMs bearing a 5-6 or 5-6-5 polycyclic system as substrates. This is the first N-methyltransferase identified in the family of PoTeMs, and the identification of this enzyme provides a new tool to generate new PoTeMs as antibiotic lead compounds.

Granulosa cells affect in vitro maturation and subsequent parthenogenetic development of buffalo (Bubalus bubalis) oocytes.

Granulosa cells (GCs) play a crucial role in follicular development and atresia. Previous studies have showed that GCs in the form of monolayer influenced in vitro maturation (IVM) of oocytes. However, the effects of GCs in the form of conditioned medium and monolayer on IVM and development competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of GC-conditioned medium (GCCM) and monolayer GC on maturation efficiency and embryo development of buffalo oocytes after parthenogenetic activation (PA). Our results showed that GCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days and 20%) exerted significant negative effects on IVM rate (41.6% vs. 44.5%), but significantly enhanced embryo development (oocyte cleavage, 81.3% vs. 69.3%; blastocyst formation, 36.3% vs. 29.3%) of buffalo oocytes after PA compared with the control group. Furthermore, monolayer GC significantly reduced both maturation efficiency (40.2% vs. 44.5%) and embryo development (oocyte cleavage, 60.6% vs. 69.3%; blastocyst formation, 20.6% vs. 29.3%) of buffalo oocytes after PA compared to the control group. Our study indicated that GCs in the form of GCCM (2 days and 20%) and monolayer GC had different effects on IVM and subsequent parthenogenetic development of buffalo oocytes.

Research on Computer-Aided Diagnosis Method Based on Symptom Filtering and Weighted Network.

In the process of disease identification, as the number of diseases increases, the collection of both diseases and symptoms becomes larger. However, existing computer-aided diagnosis systems do not completely solve the dimensional disaster caused by the increasing data set. To address the above problems, we propose methods of using symptom filtering and a weighted network with the goal of deeper processing of the collected symptom information. Symptom filtering is similar to a filter in signal transmission, which can filter the collected symptom information, further reduce the dimensional space of the system, and make the important symptoms more prominent. The weighted network, on the other hand, mines deeper disease information by modeling the channels of symptom information, amplifying important information, and suppressing unimportant information. Compared with existing hierarchical reinforcement learning models, the feature extraction methods proposed in this paper can help existing models improve their accuracy by more than 10%.

The coupled reaction catalyzed by EchB and EchC lead to the formation of the common 2',3',5'-trihydroxy-benzene core in echosides biosynthesis.

p-Terphenyls represent a unique family of aromatic natural products generated by nonribosomal peptide synthetase-like (NRPS-like) enzyme. After formation of p-terphenyl skeleton, tailoring modifications will give rise to structural diversity and various biological activities. Here we demonstrated a two-enzyme (EchB, a short-chain dehydrogenase/reductase (SDR), and EchC, a nuclear transport factor 2 (NTF2)-like dehydratase) participated transformation from dihydroxybenzoquinone core to 2',3',5'-trihydroxy-benzene in the biosynthesis of echosides. Beginning with polyporic acid as substrate, successive steps of reduction-dehydration-reduction cascade catalyzed by EchB-EchC-EchB were concluded after in vivo gene disruption and in vitro bioassay experiments. These findings demonstrated a conserved synthesis pathway of 2',3',5'-trihydroxy-p-terphenyls in bacteria, such as Actinomycetes and Burkholderia. The parallel pathway in fungi has yet to be explored.

Targeted Discovery of the Polyene Macrolide Hexacosalactone A from Streptomyces by Reporter-Guided Selection of Fermentation Media.

New approaches are still needed to fully explore the biosynthetic potential of microbes. We recently devised a melC reporter-guided fermentation media screening approach for targeted activation of cryptic gene clusters. Using this approach, we successfully activated the expression of the hcl gene cluster in Streptomyces sp. LZ35 and discovered a novel polyene macrolide hexacosalactone A (1).

Expression of the Clifednamide Biosynthetic Pathway in Streptomyces Generates 27,28-seco-Derivatives.

Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP natural products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the cytochrome P450 enzyme CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.

OX4 Is an NADPH-Dependent Dehydrogenase Catalyzing an Extended Michael Addition Reaction To Form the Six-Membered Ring in the Antifungal HSAF.

The polycyclic tetramate macrolactam HSAF is an antifungal natural product isolated from Lysobacter enzymogenes. HSAF and its analogues have a distinct chemical structure and new mode of antifungal action. The mechanism by which the 5/5/6 tricycle of HSAF is formed from the polyene precursor is not totally clear. Here, we used purified OX4, a homologous enzyme of alcohol dehydrogenase/Zn-binding proteins, to show the enzymatic mechanism for six-membered ring formation. The results from the deuterium isotope incorporation demonstrated that OX4 selectively transfers the pro-R hydride of NADPH to C21 and one proton from water to C10 of 3-deOH alteramide C (1), resulting in 3-deOH HSAF (2) through a reductive cyclization of the polyene precursor by a mechanism consistent with an extended 1,6-Michael addition reaction. The regioselective incorporation of the NADPH hydride into C21 of 1 is also stereoselective, leading to the 21S configuration of 2. This work represents the first characterization of the activity and selectivity of the enzyme for six-membered ring formation in a group of distinct antifungal polycyclic tetramate macrolactams.

Biosynthesis of the Polycyclic System in the Antifungal HSAF and Analogues from Lysobacter enzymogenes.

The biocontrol agent Lysobacter enzymogenes produces polycyclic tetramate macrolactams (PoTeMs), including the antifungal HSAF. To elucidate the biosynthesis of the cyclic systems, we identified eleven HSAF precursors/analogues with zero, one, two, or three rings through heterologous expression of the HSAF gene cluster. A series of combinatorial gene expression and deletion experiments showed that OX3 is the "gatekeeper" responsible for the formation of the first 5-membered ring from lysobacterene A, OX1 and OX2 are responsible for formation of the second ring but with different selectivity, and OX4 is responsible for formation of the 6-membered ring. In vitro experiments showed that OX4 is an NADPH-dependent enzyme that catalyzes the reductive cyclization of 3-dehydroxy alteramide C to form 3-dehydroxy HSAF. Thus, the multiplicity of OX genes is the basis for the structural diversity of the HSAF family, which is the only characterized PoTeM cluster that involves four redox enzymes in the formation of the cyclic system.

Alteramide B is a microtubule antagonist of inhibiting Candida albicans.

BACKGROUND: Alteramide B (ATB), isolated from Lysobacter enzymogenes C3, was a new polycyclic tetramate macrolactam (PTM). ATB exhibited potent inhibitory activity against several yeasts, particularly Candida albicans SC5314, but its antifungal mechanism is unknown. METHODS: The structure of ATB was established by extensive spectroscopic analyses, including high-resolution mass spectrometry, 1D- and 2D-NMR, and CD spectra. Flow cytometry, fluorescence microscope, transmission electron microscope, molecular modeling, overexpression and site-directed mutation studies were employed to delineate the anti-Candida molecular mechanism of ATB. RESULTS: ATB induced apoptosis in C. albicans through inducing reactive oxygen species (ROS) production by disrupting microtubules. Molecular dynamics studies revealed the binding patterns of ATB to the ß-tubulin subunit. Overexpression of the wild type and site-directed mutants of the ß-tubulin gene (TUBB) changed the sensitivity of C. albicans to ATB, confirming the binding of ATB to ß-tubulin, and indicating that the binding sites are L215, L217, L273, L274 and R282. In vivo, ATB significantly improved the survival of the candidiasis mice and reduced fungal burden. CONCLUSION: The molecular mechanism underlying the ATB-induced apoptosis in C. albicans is through inhibiting tubulin polymerization that leads to cell cycle arrest at the G2/M phase. The identification of ATB and the study of its activity provide novel mechanistic insights into the mode of action of PTMs against the human pathogen. GENERAL SIGNIFICANCE: This study shows that ATB is a new microtubule inhibitor and a promising anti-Candida lead compound. The results also support ß-tubulin as a potential target for anti-Candida drug discovery.

In vitro reconstitution of a PKS pathway for the biosynthesis of galbonolides in Streptomyces sp. LZ35.

The galbonolides are 14-membered macrolide antibiotics with a macrocyclic backbone similar to that of erythromycins. Galbonolides exhibit broad-spectrum antifungal activities. Retro-biosynthetic analysis suggests that the backbone of galbonolides is assembled by a type I modular polyketide synthase (PKS). Unexpectedly, the galbonolide biosynthetic gene cluster, gbn, in Streptomyces sp. LZ35 encodes a hybrid fatty acid synthase (FAS)-PKS pathway. In vitro reconstitution revealed the functions of GbnA (an AT-ACP didomain protein), GbnC (a FabH-like enzyme), and GbnB (a novel multidomain PKS module without AT and ACP domains) responsible for assembling the backbone of galbonolides, respectively. To our knowledge, this study is the first biochemical characterization of a hybrid FAS-PKS pathway for the biosynthesis of 14-membered macrolides. The identification of this pathway provides insights into the evolution of PKSs and could facilitate the design of modular pools for synthetic biology.

Identification of AstG1, A LAL family regulator that positively controls ansatrienins production in Streptomyces sp. XZQH13.

Ansamycins is a group of type I polyketides characterized by the unique starter unit 3-amino-5-hydroxybenzoic acid. This family of secondary metabolites shows diverse biological activities, well-known members of which include rifamycin, geldanamycin, and maytansine. Previously, we isolated an AHBA synthase gene-positive strain Streptomyces sp. XZQH13 containing a "silent" ansamycin biosynthetic gene cluster ast. The constitutive expression of the Large-ATP-binding regulators of the LuxR family regulator gene astG1 located within the cluster triggered the expression of the biosynthetic genes. Reverse transcription-PCR experiments showed that the expression of the key biosynthetic genes, astB4, astD1, and astF1, was induced in the astG1 overexpression mutant compared to the wild type. This led to the isolation of two known ansatrienins, hydroxymycotrienin A (1) and thiazinotrienomycin G (2), which were identified by analysis of the mass spectral and NMR spectral data, from the mutant. These observations suggest that astG1 is probably a pathway-specific positive regulator for the biosynthesis of ansatrienin.

Biosynthesis of hygrocins, antitumor naphthoquinone ansamycins produced by Streptomyces sp. LZ35.

Hygrocins are naphthoquinone ansamycins with significant antitumor activities. Here, we report the identification and characterization of the hygrocin biosynthetic gene cluster (hgc) in Streptomyces sp. LZ35. A biosynthetic pathway is proposed based on bioinformatics analysis of the hgc genes and intermediates accumulated in selected gene disruption mutants. One of the steps during the biosynthesis of hygrocins is a Baeyer­Villiger oxidation between C5 and C6, catalyzed by luciferase- like monooxygenase homologue Hgc3. Hgc3 represents the founding member of a previously uncharacterized family of enzymes acting as Baeyer­Villiger monooxygenases.

Iterative assembly of two separate polyketide chains by the same single-module bacterial polyketide synthase in the biosynthesis of HSAF.

Antifungal HSAF (heat-stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two "clean" strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl-coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene-PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS-NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide-peptide natural products.

Enhancing the Performance of Perovskite Solar Cells via the Functional Group Synergistic Effect in Interfacial Passivation Materials.

Interfacial defects are considered to be a stumbling block in producing highly efficient perovskite solar cells (PSCs), so a more reasonable design is required for interfacial passivation materials (IPMs) to achieve further improvements in PSC performance. Here, we use fluorine atom (-F) and methoxy (-OCH3) functional groups to modify the same molecular fragment, obtaining three kinds of IPMs named YZ-301, YZ-302, and YZ-303, respectively. Through the subtle combination of -F and -OCH3, the fragment in YZ-302 exhibits an enhanced electronegativity, rendering the correlative IPM with a stronger interaction with the perovskite layer. As a result, YZ-302 shows the best defect passivation and hole transport effect at the interface, and the PSC based on YZ-302 treatment achieves the best efficiency approaching 24%, which is better than the reference and devices with other IPMs, and it also has excellent device stability.

A pooling convolution model for multi-classification of ECG and PCG signals.

Electrocardiogram (ECG) and phonocardiogram (PCG) signals are physiological signals generated throughout the cardiac cycle. The application of deep learning techniques to recognize ECG and PCG signals can greatly enhance the efficiency of cardiovascular disease detection. Therefore, we propose a series of straightforward and effective pooling convolutional models for the multi-classification of ECG and PCG signals. Initially, these signals undergo preprocessing. Subsequently, we design various structural blocks, including a stacked block (MCM) comprising convolutional layer and max-pooling layers, along with its variations, as well as a residual block (REC). By adjusting the number of structural blocks, these models can handle ECG and PCG data with different sampling rates. In the final tests, the models utilizing the MCM structural block achieved accuracies of 98.70 and 92.58% on the ECG and PCG fusion datasets, respectively. These accuracies surpass those of all networks utilizing its variations. Moreover, compared to the models employing the REC structural block, the accuracies are improved by 0.02 and 4.30%, respectively. Furthermore, this research has been validated through tests conducted on multiple ECG and PCG datasets, along with comparisons to other published literature. To further validate the generalizability of the model, an additional experiment involving the classification of a synchronized ECG-PCG dataset was conducted. This dataset is divided into seven different levels of fatigue based on the amount of exercise performed by each healthy subject during the testing process. The results indicate that the model using the MCM block also achieved the highest accuracy.