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With the emergence of drug-resistant strains, the treatment of tuberculosis (TB) is becoming more difficult and there is an urgent need to find new anti-TB drugs. Mycobacterium marinum, as a model organism of Mycobacterium tuberculosis, can be used for the rapid and efficient screening of bioactive compounds. The 14-membered resorcylic acid lactones (RALs) have a wide range of bioactivities such as antibacterial, antifouling and antimalarial activity. In order to further study their bioactivities, we initially constructed a 14-membered RALs library, which contains 16 new derivatives. The anti-M. marinum activity was evaluated in vitro. Derivatives 12, 19, 20 and 22 exhibited promising activity with MIC90 values of 80, 90, 80 and 80 μM, respectively. The preliminary structure–activity relationships showed that the presence of a chlorine atom at C-5 was a key factor to improve activity. Further studies showed that 12 markedly inhibited the survival of M. marinum and significantly reduced the dosage of positive drugs isoniazid and rifampicin when combined with them. These results suggest that 12 is a bioactive compound capable of enhancing the potency of existing positive drugs, and its effective properties make it a very useful leads for future drug development in combating TB resistance.

Despite numerous studies on transcriptional level regulation by single genes in drug producing Actinomyces, the global regulation based on epigenetic modification is not well explored. N4-methylcytosine (m4C), an abundant epigenetic marker in Actinomycetes’ genome, but its regulatory mechanism remains unclear. In this study, we identify a m4C methyltransferase (SroLm3) in Streptomyces roseosporus L30 and multi-omics studies were performed and revealed SroLm3 as a global regulator of secondary metabolism. Notably, three BGCs in ΔsroLm3 strain exhibited decreased expression compared to wild type. In-frame deletion of sroLm3 in S.roseosporus L30 further revealed its role in enhancing daptomycin production. In summary, we characterized a m4C methyltransferase, revealed the function of m4C in secondary metabolism regulation and biosynthesis of red pigment, and mapped a series of novel regulators for daptomycin biosynthesis dominated by m4C methylation. Our research further indicated that m4C DNA methylation may contribute to a metabolic switch from primary to secondary metabolism in Actinomyces.

Multidrug resistance (MDR) is the main cause of clinical treatment failure and poor prognosis in cancer. Targeting P-glycoprotein (P-gp) has been regarded as an effective strategy to overcome MDR. In this work, we reported our preclinical studies of the triazolo[1,5-a]pyrimidine-based compound WS-716 as a highly potent, specific, and orally active P-gp inhibitor. Through direct binding to P-gp, WS-716 inhibited efflux function of P-gp and specifically reversed P-gp-mediated MDR to paclitaxel (PTX) in multiple resistant cell lines, without changing its expression or subcellular localization. WS-716 and PTX synergistically inhibited formation of colony and 3D spheroid, induced apoptosis and cell cycle arrest at G2/M phase in resistant SW620/Ad300 cells. In addition, WS-716 displayed minimal effect on the drug-metabolizing enzyme cytochrome P4503A4 (CYP3A4). Importantly, WS-716 increased sensitivity of both pre-clinically and clinically derived MDR tumors to PTX in vivo with the T/C value of 29.7% in patient-derived xenograft (PDX) models. Relative to PTX treatment alone, combination of WS-716 and PTX caused no obvious adverse reactions. Taken together, our preclinical studies revealed therapeutic promise of WS-716 against MDR cancer, the promising data warrant its further development for cancer therapy.

ABSTRACT DNA methylation is a defense that microorganisms use against extreme environmental stress, and improving resistance against environmental stress is essential for industrial actinomycetes. However, research on strain optimization utilizing DNA methylation for breakthroughs is rare. Based on DNA methylome analysis and KEGG pathway assignment in Streptomyces roseosporus, we discovered an environmental stress resistance regulator, TagR. A series of in vivo and in vitro experiments identified TagR as a negative regulator, and it is the first reported regulator of the wall teichoic acid (WTA) ABC transport system. Further study showed that TagR had a positive self-regulatory loop and m4C methylation in the promoter improved its expression. The ΔtagR mutant exhibited better hyperosmotic resistance and higher decanoic acid tolerance than the wild type, which led to a 100% increase in the yield of daptomycin. Moreover, enhancing the expression of the WTA transporter resulted in better osmotic stress resistance in Streptomyces lividans TK24, indicating the potential for wide application of the TagR-WTA transporter regulatory pathway. This research confirmed the feasibility and effectiveness of mining regulators of environmental stress resistance based on the DNA methylome, characterized the mechanism of TagR, and improved the resistance and daptomycin yield of strains. Furthermore, this research provides a new perspective on the optimization of industrial actinomycetes. IMPORTANCE This study established a novel strategy for screening regulators of environmental stress resistance based on the DNA methylome and discovered a new regulator, TagR. The TagR-WTA transporter regulatory pathway improved the resistance and antibiotic yield of strains and has the potential for wide application. Our research provides a new perspective on the optimization and reconstruction of industrial actinomycetes.

Vortioxetine is currently used as the first-line therapy drug for major depressive disorder (MDD). In the present study, we aimed to develop and fully validate an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of vortioxetine and its major metabolite Lu AA34443 in plasma and to investigate the effects of dronedarone and amiodarone on vortioxetine metabolism in rats. After protein precipitation with acetonitrile, the separation of vortioxetine, Lu AA34443 and duloxetine (internal standard, IS) were finished on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column and their detections were conducted by a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion mode. The assay displayed excellent linearity in the range of 0.5–50 ng/mL for vortioxetine, and 5–1000 ng/mL for Lu AA34443. The results of this method exhibited that the precision, accuracy, matrix effect, recovery, and stability of vortioxetine and Lu AA34443 met all requirements for the quantitation in plasma samples. The validated assay was further successfully employed to study the effects of dronedarone (80 mg/kg) and amiodarone (60 mg/kg) on vortioxetine metabolism in rats. The results showed that dronedarone and amiodarone could increase the concentration of vortioxetine and have inhibitory effect on vortioxetine metabolism. Thus, vortioxetine dose interruption or reduction may be considered.

Daptomycin is a cyclic lipopeptide antibiotic with a significant antibacterial action against antibiotic-resistant Gram-positive bacteria. Despite numerous attempts to enhance daptomycin yield throughout the years, the production remains unsatisfactory. This study reports the application of multilevel metabolic engineering strategies in Streptomyces roseosporus to reconstruct high-quality daptomycin overproducing strain L2797-VHb, including precursor engineering (i.e., refactoring kynurenine pathway), regulatory pathway reconstruction (i.e., knocking out negative regulatory genes arpA and phaR), byproduct engineering (i.e., removing pigment), multicopy biosynthetic gene cluster (BGC), and fermentation process engineering (i.e., enhancing O2 supply). The daptomycin titer of L2797-VHb arrived at 113 mg/l with 565% higher comparing the starting strain L2790 (17 mg/l) in shake flasks and was further increased to 786 mg/l in 15 L fermenter. This multilevel metabolic engineering method not only effectively increases daptomycin production, but can also be applied to enhance antibiotic production in other industrial strains.

BackgroundTo evaluate the prognostic value of serum inflammatory biomarkers and develop a risk stratification model for high-grade glioma (HGG) patients based on clinical, laboratory, radiological, and pathological factors.Materials and MethodsA retrospective study of 199 patients with HGG was conducted. Patients were divided into a training cohort (n = 120) and a validation cohort (n = 79). The effects of potential associated factors on the overall survival (OS) time were investigated and the benefits of serum inflammatory biomarkers in improving predictive performance was assessed. Univariable and multivariable Cox regression analyses, the least absolute shrinkage and selection operator (LASSO) regression analysis, and support vector machines (SVM) were used to select variables for the final nomogram model.ResultsAfter multivariable Cox, LASSO, and SVM analysis, in addition to 3 other clinico-pathologic factors, platelet-to-lymphocyte ratio (PLR) >144.4 (hazard ratio [HR], 2.05; 95% confidence interval [CI], 1.25–3.38; P = 0.005) were left for constructing the predictive model. The model with PLR exhibited a better predictive performance than that without them in both cohorts. The nomogram based on the model showed an excellent ability of discrimination in the entire cohort (C-index, 0.747; 95%CI, 0.706–0.788). The calibration curves showed good consistency between the predicted and observed survival probability.ConclusionOur study confirmed the prognostic value of serum inflammatory biomarkers including PLR and established a comprehensive scoring system for the OS prediction in HGG patients.

Abstract Background Anthurium andraeanum, an important ornamental flower, has to go through a growth-delaying period after transfer from tissue culture to soil, which requires time and extra costs. Furthermore, during this period, the plantlets are highly susceptible to bacterial infections, which results in impaired development and severe losses. Here, we aimed to address whether application of the endophytic fungus, Piriformospora indica protects the A. andraeanum root system during the critical propagation period, and whether P. indica reduce the mortality rate by stimulating the host’s resistance against diseases. Results We demonstrate that P. indica shortens the recovery period of Anthurium, promotes growth and confers disease resistance. The beneficial effect of P. indica results in faster elongation of Anthurium roots early in the interaction. P. indica-colonized plants absorb more phosphorus and exhibit higher photosynthesis rates than uncolonized control plants. Moreover, higher activities of stress-related enzymes, of jasmonic acid levels and mRNA levels of jasmonic acid-responsive genes suggest that the fungus prepares the plant to respond more efficiently to potentially upcoming threats, including bacterial wilt. Conclusion These results suggest that P. indica is a helpful symbiont for promoting Anthurium rooting and development. All our evidences are sufficient to support the disease resistance conferred by P. indica through the plant-fungal symbiosis. Furthermore, it implicates that P. indica has strong potential as bio-fertilizer for utilization in ornamental plant cultivation.

Abstract Two new cyclopentapeptides, xylapeptide A (1) with an uncommon L-pipecolinic acid moiety, and xylapeptide B (2) having a common L-proline residue were identified from an associated fungus Xylaria sp. isolated from the Chinese medicinal plant Sophora tonkinensis. Their planar structures were elucidated by a comprehensive analysis of NMR and MS spectroscopic spectra. The absolute configurations were determined by Marfey’s method and single-crystal X-ray diffraction (Cu Kα) analysis. Xylapeptide A (1) is the first example of cyclopentapeptide with L-Pip of terrestrial origin and showed strong antibacterial activity against Bacillus subtilis and B. cereus with MIC value of 12.5 μg/mL.

Three new diketopiperazine alkaloids, including two oxepine-containing diketopiperazines, chrysopiperazines A and B (1 and 2), and one quinazoline-containing diketopiperazine, chrysopiperazine C (5), together with three known analogues (3, 4, and 6), were isolated from the gorgonian-derived Penicillium chrysogenum fungus. The relative and absolute configurations of C-3 and C-15 in 1 and 2, C-3 and C-14 in 5 were established by NOE modified Marfey’s analysis and electronic circular dichroism (ECD) calculations. Particularly, the absolute configurations of C-19 in 1 and 3, which was very challenging to be identified due to the flexible conformation in a short aliphatic chain, were successfully determined by the vibrational circular dichroism (VCD) method, supplying with a reliable and optional method to define the absolute configurations. Additionally, this is the first report on oxepine-containing diketopiperazines from the genus Penicillium.

One new hydroanthraquinone dimer with a rare C-9–C-7′ linkage, nigrodiquinone A (1), and four known anthraquinone monomers 2–5, were isolated from a fungus Nigrospora sp. obtained from the zoanthid Palythoa haddoni collected in the South China Sea. The structure of 1 was established through extensive NMR spectroscopy, and the absolute configuration was elucidated by comparing computed electronic circular dichroism (ECD) and optical rotations (OR) with experimental results. All the compounds were evaluated for antiviral activity, and 1 was also evaluated for antibacterial activity. Compound 4 displayed mild antiviral activity against coxsackie virus (Cox-B3) with the IC50 value of 93.7 μM, and 5 showed an IC50 value of 74.0 μM against respiratory syncytial virus (RSV).

We say that a space $X$ has the discrete countable chain condition (DCCC for short) if every discrete family of nonempty open subsets of $X$ is countable. A space $X$ has a zeroset diagonal if there is a continuous mapping $f X^2 \rightarrow[0,1]$ with $\Delta_X=f^{-1}(0)$, where $\Delta_X=\{(x,x)\colon x\in X\}$. In this paper, we prove that every first countable DCCC space with a zeroset diagonal has cardinality at most $\mathfrak c$.

The dynamics by which polymeric protein filaments divide in the presence of negligible growth, for example due to the depletion of free monomeric precursors, can be described by the universal mathematical equations of 'pure fragmentation'. The rates of fragmentation reactions reflect the stability of the protein filaments towards breakage, which is of importance in biology and biomedicine for instance in governing the creation of amyloid seeds and the propagation of prions. Here, we devised from mathematical theory inversion formulae to recover the division rates and division kernel information from time-dependent experimental measurements of filament size distribution. The numerical approach to systematically analyze the behaviour of pure fragmentation trajectories was also developed. We illustrate how these formulae can be used, provide some insights on their robustness, and show how they inform the design of experiments to measure fibril fragmentation dynamics. These advances are made possible by our central theoretical result on how the length distribution profile of the solution to the pure fragmentation equation aligns with a steady distribution profile for large times.

We study relationships between separability with other properties in semi-stratifiable spaces. Especially, we prove the following statements: (1) If $X$ is a semi-stratifiable space, then $X$ is separable if and only if $X$ is $DC(ømega_1)$; (2) If $X$ is a star countable extent semi-stratifiable space and has a dense metrizable subspace, then $X$ is separable; (3) Let $X$ be a $ømega$-monolithic star countable extent semi-stratifiable space. If $t(X)=ømega$ and $d(X) \leømega_1$, then $X$ is hereditarily separable. Finally, we prove that for any $T_1$-space $X$, $|X| \le L(X)^{\Delta(X)}$, which gives a partial answer to a question of Basile, Bella, and Ridderbos (2011). As a corollary, we show that $|X| \le e(X)^{ømega}$ for any semi-stratifiable space $X$.

The division of amyloid fibril particles through fragmentation is implicated in the progression of human neurodegenerative disorders such as Parkinson’s disease. Fragmentation of amyloid fibrils plays a crucial role in the propagation of the amyloid state encoded in their three-dimensional structures and may have an important role in the spreading of potentially pathological properties and phenotypes in amyloid-associated diseases. However, despite the mechanistic importance of fibril fragmentation, the relative stabilities of different types or different polymorphs of amyloid fibrils toward fragmentation remain to be quantified. We have previously developed an approach to compare the relative stabilities of different types of amyloid fibrils toward fragmentation. In this study, we show that controlled sonication, a widely used method of mechanical perturbation for amyloid seed generation, can be used as a form of mechanical perturbation for rapid comparative assessment of the relative fragmentation stabilities of different amyloid fibril structures. This approach is applied to assess the relative fragmentation stabilities of amyloid formed in vitro from wild type (WT) α-synuclein and two familial mutant variants of α-synuclein (A30P and A53T) that generate morphologically different fibril structures. Our results demonstrate that the fibril fragmentation stabilities of these different α-synuclein fibril polymorphs are all highly length dependent but distinct, with both A30P and A53T α-synuclein fibrils displaying increased resistance towards sonication-induced fibril fragmentation compared with WT α-synuclein fibrils. These conclusions show that fragmentation stabilities of different amyloid fibril polymorph structures can be diverse and suggest that the approach we report here will be useful in comparing the relative stabilities of amyloid fibril types or fibril polymorphs toward fragmentation under different biological conditions.

Summary: The division of amyloid protein fibrils is required for the propagation of the amyloid state and is an important contributor to their stability, pathogenicity, and normal function. Here, we combine kinetic nanoscale imaging experiments with analysis of a mathematical model to resolve and compare the division stability of amyloid fibrils. Our theoretical results show that the division of any type of filament results in self-similar length distributions distinct to each fibril type and the conditions applied. By applying these theoretical results to profile the dynamical stability toward breakage for four different amyloid types, we reveal particular differences in the division properties of disease-related amyloid formed from α-synuclein when compared with non-disease associated model amyloid, the former showing lowered intrinsic stability toward breakage and increased likelihood of shedding smaller particles. Our results enable the comparison of protein filaments' intrinsic dynamic stabilities, which are key to unraveling their toxic and infectious potentials.

A topological space $X$ is said to be star Lindelöf if for any open cover $\mathcal U$ of $X$ there is a Lindelöf subspace $A \subset X$ such that $øperatorname{St}(A, \mathcal U)=X$. The "extent" $e(X)$ of $X$ is the supremum of the cardinalities of closed discrete subsets of $X$. We prove that under $V=L$ every star Lindelöf, first countable and normal space must have countable extent. We also obtain an example under ${\rm MA +\nobreak\neg CH$, which shows that a star Lindelöf, first countable and normal space may not have countable extent.}

A topological space $X$ has a rank 2-diagonal if there exists a diagonal sequence on $X$ of rank $2$, that is, there is a countable family $\{\mathcal U_n n\inømega\}$ of open covers of $X$ such that for each $x \in X$, $\{x\}=\bigcap\{{\rm St}^2(x, \mathcal U_n) n \inømega\}$. We say that a space $X$ satisfies the Discrete Countable Chain Condition (DCCC for short) if every discrete family of nonempty open subsets of $X$ is countable. We mainly prove that if $X$ is a DCCC normal space with a rank 2-diagonal, then the cardinality of $X$ is at most $\mathfrak c$. Moreover, we prove that if $X$ is a first countable DCCC normal space and has a $G_\delta$-diagonal, then the cardinality of $X$ is at most $\mathfrak c$.

Transmissible amyloid particles called prions are associated with infectious prion diseases in mammals and inherited phenotypes in yeast. All amyloid aggregates can give rise to potentially infectious seeds that accelerate their growth. Why some amyloid seeds are highly infectious prion particles while others are less infectious or even inert, is currently not understood. To address this question, we analyzed the suprastructure and dimensions of synthetic amyloid fibrils assembled from the yeast (Saccharomyces cerevisiae) prion protein Sup35NM. We then quantified the ability of these particles to induce the [PSI+] prion phenotype in cells. Our results show a striking relationship between the length distribution of the amyloid fibrils and their ability to induce the heritable [PSI+] prion phenotype. Using a simple particle size threshold model to describe transfection activity, we explain how dimensions of amyloid fibrils are able to modulate their infectious potential as prions.

Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy. Salt was found to induce an increase in β-hairpin structure for the C-terminal fragment (residues 41 – 56), whereas no major salt effect on structure was observed for the isolated N-terminal fragment (residues 1 – 41). In line with the increasing evidence on the interrelation between fragment complementation and stability of the corresponding intact protein, we also find that salt effects on reconstitution can be predicted from salt dependence of the stability of the intact protein. Our data show that our variant (which has the mutations T2Q, N8D, N37D and reconstitutes in a manner similar to the wild type) displays the lowest equilibrium association constant around physiological salt concentration, with higher affinity observed both at lower and higher salt concentration. This corroborates the salt effects on the stability towards denaturation of the intact protein, for which the stability at physiological salt is lower compared to both lower and higher salt concentrations. Hence we conclude that reconstitution reports on molecular factors that govern the native states of proteins.

There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity.

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of β2-microglobulin (β2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which β2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of β2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that β2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between β2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of β2m amyloid-associated osteoarticular tissue destruction in DRA.

Abstract Background Low-molecular-weight chitosan oligosaccharide (LMCOS), a chitosan degradation product, is water-soluble and easily absorbable, rendering it a popular biomaterial to study. However, its effect on bone remodelling remains unknown. Therefore, we evaluated the effect of LMCOS on lipopolysaccharide (LPS)-induced bone resorption in mice. Methods Six-week-old male C57BL/6 mice (n = five per group) were randomly divided into five groups: PBS, LPS, LPS + 0.005% LMCOS, LPS + 0.05% LMCOS, and LPS + 0.5% LMCOS. Then, the corresponding reagents (300 μL) were injected into the skull of the mice. To induce bone resorption, LPS was administered at 10 mg/kg per injection. The mice were injected three times a week with PBS alone or LPS without or with LMCOS and sacrificed 2 weeks later. The skull was removed for micro-computed tomography, haematoxylin-eosin staining, and tartrate-resistant acid phosphatase staining. The area of bone damage and osteoclast formation were evaluated and recorded. Results LMCOS treatment during LPS-induced skull resorption led to a notable reduction in the area of bone destruction; we observed a dose-dependent decrease in the area of bone destruction and number of osteoclasts with increasing LMCOS concentration. Conclusions Our findings showed that LMCOS could inhibit skull bone damage induced by LPS in mice, further research to investigate its therapeutic potential for treating osteolytic diseases is required.